Pregnancy up-regulated, nonubiquitous CaM kinase

ABSTRACT

This invention relates generally to a novel CaM multi-functional protein kinase, which has been named Pregnancy Up-Regulated, Nonubiquitous CaM Kinase (PNCK), and to the nucleotide sequence encoding it. The kinase is temporally expressed during postnatal mammary development in a spatially heterogeneous manner in certain subsets of cells, and overexpressed in a subset of primary breast cancers. The invention further relates to an analysis of a correlation between carcinogenesis and postnatal development, particularly mammary development, especially associated with parity; as well as to methods of using the kinase, or gene encoding it, as markers, prognostic tools, screening tools and therapies, in vitro and in vivo that are based upon that correlation.

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application60/257,073 filed Dec. 21, 2000.

GOVERNMENT SUPPORT

This work was supported in part by grants from the Elsa U. PardeeFoundation, Grant RPG-99-259-01-DDC from the American Cancer Society,the National Institutes of Health Grants CA83849, CA71513, and CA78410from the National Cancer Institute, and United States Army Breast CancerResearch Program Grants DAMD17-96-1-6112, DAMD17-98-1-8226,DAMD-99-1-9463, and DAMD-99-1-9349.

FIELD OF THE INVENTION

This invention relates generally to a novel CaM multi-functional proteinkinase, specifically to Pregnancy Up-Regulated, Nonubiquitous CaM Kinase(PNCK), which is temporally expressed in mammary development in aspatially heterogeneous manner in certain subsets of cells, andoverexpressed in a subset of primary breast cancers; and to analysis ofa correlation between carcinogenesis and postnatal development,particularly mammary development, especially associated with parity.

BACKGROUND

Numerous epidemiologic and animal studies analyzing the impact ofreproductive events such as puberty, pregnancy, and parity on earlyevents in carcinogenesis suggest that the developmental state of thebreast plays a critical role in the determination of breast cancer risk(Lambe et al., N. Engl. J. Med., 331:5–9 (1994); MacMahon et al., Bull.WHO, 43:209–221 (1970); MacMahon et al., Int. J. Cancer, 29:13–16(1982); Newcomb et al., N. Engl. J. Med., 330:81–87 (1994); Russo etal., J. Natl. Cancer Inst., 61:1439–1449 (1978); Russo et al., Lab.Invest., 57:112–137 (1987)). In fact, a woman's lifetime risk ofdeveloping breast cancer is intrinsically related to reproductiveevents, particularly those that affect the differentiated state of thebreast. Results from both human epidemiology and animal model systemsindicate that an early first full-term pregnancy results in a permanentchange in the breast that confers a decreased risk for the subsequentdevelopment of breast cancer (Medina et al, J. Natl. Cancer Inst.,91:967–969 (1999); Russo et al., Breast Cancer Res. Treat., 2:5–73,(1982); Russo et al., J. Natl. Cancer Inst., 61:1439–1449 (1978);MacMahon et al, 1970). This implies an intrinsic relationship betweenthe process of carcinogenesis and normal pathways of differentiation anddevelopment in the breast.

The findings that aborted pregnancies, the majority of which occur priorto the third trimester, are not protective against breast cancer andthat lactation has only a minimal protective effect compared withfull-term pregnancy suggest that parity-induced protection againstbreast cancer results from physiological changes that occur late inpregnancy (Michels et al., Cancer Causes Control, 6:75–82 (1995); Melbyeet al., N. Engl. J. Med., 336:81–85 (1997)). As a result, the protectiveeffect of parity has been hypothesized to result from the impact ofterminal differentiation on the susceptibility of the mammary epitheliumto carcinogenesis (Russo et al., 1982; Russo et al., 1978).Nevertheless, the molecular and cellular basis for this phenomenon isunknown. As such, understanding the developmental changes that occur inthe breast late in pregnancy is essential for understanding theprotected state of the breast associated with parity, particularly withrespect to genes that control mammary proliferation and differentiation.

Protein kinases represent the largest class of genes known to regulatedifferentiation, development, and carcinogenesis in eukaryotes. Manyprotein kinases function as intermediates in signal transductionpathways that control complex processes such as differentiation,development, and carcinogenesis (Birchmeier et al., BioEssays,15:185–190 (1993); Bolen, Oncogene, 8:2025–2031 (1993); Rawlings et al.,Immunol. Rev., 138:105–119 (1994)). Accordingly, studies of proteinkinases in a wide range of biological systems have led to a morecomprehensive understanding of the regulation of cell growth anddifferentiation (Bolen 1993; Fantl et al., Annu. Rev. Biochem.,62:453–481(1993); Hardie, Symp. Soc. Exp. Biol., 44:241–255 (1990)).

Not surprisingly, several members of the protein kinase family have beenreported to be involved in the pathogenesis of cancer both in humans andin rodent model systems (Cardiff et al., Cancer Surv., 16:97–113(1993);Dickson et al., Cancer Treatment Res., 61:249–273 (1992); Guy et al.,Genes Dev., 8:23–32 (1994); Guy et al., Proc. Natl. Acad. Sci. USA,89:10578–10582 (1992); Slamon et al., Science, 244:707–712 (1989)). Forinstance, the EGF receptor and ErbB2/HER2 are each amplified andoverexpressed in subsets of highly aggressive breast cancers, and thesemolecules may thereby provide prognostic information relevant toclinical treatment and outcome (Klijn et al., Cold Spring HarborLaboratory Press, Vol. 18, pp. 165–198 (1993); Slamone et al., Science,235, 177–182 (1987); Slamon et al., 1989). Furthermore, overexpressionof specific protein kinases, or of ligands for protein kinases, in themammary epithelium of transgenic animals results in neoplastictransformation (Cardiff et al., 1993); Guy et al., 1994); Muller et al.,Cell, 54:105–115 (1988) Muller et al., EMBO J., 9:907–913 (1990)).

One particular family of protein kinases, the Ca²⁺/calmodulin-dependent(CaM) kinases are known to regulate cellular processes as diverse asneurotransmitter release, muscle contraction, cell cycle control,transcriptional regulation, metabolism, and gene transcription (Fukunagaet al., Jpn. J. Pharmacol., 79:7–15 (1999); Matthews et al., Mol. Cell.Biol., 14:6107–6116 (1994); Polishchuk et al., FEBS Lett., 362:271–275(1995); Schulman, Curr. Opin. Cell Biol., 5:247–253 (1993); Sheng et al,Science, 252:1427–1430 (1991)). For example, point mutations in theDrosophila calmodulin gene result in defects in development includingpupal lethality and ectopic wing vein formation (Nelson et al.,Genetics, 147:1783–1798 (1997)). Furthermore, calmodulin expression isregulated during cardiac development, and overexpression of calmodulinin murine cardiomyocytes results in cardiomyocyte hypertrophy (Gruver etal., Endocrinology, 133:376–388 (1993)). Like calmodulin, it has beenreported that CaM kinases play diverse roles in development includingCaMKIV in T-cell maturation and CaMKII in cell cycle regulation (Lukaset al., San Diego: Academic Press 65–168 (1998); Nairn et al., Semin.Cancer Biol., 5:295–303 (1994); Hanissian et al., J. Biol. Chem.,268:20055–20063 (1993); Krebs et al., Biochem. Biophys. Res. Commun.,241:383–389(1997)).

Ca²⁺ is an important intracellular second-messenger molecule ineukaryotic signal transduction pathways. Many of the effects of Ca²⁺ aremediated through its interaction with the Ca²⁺-binding protein,calmodulin. The Ca²⁺/calmodulin complex is, in turn, required formaximal activation of CaM-dependent protein kinases. In addition, as afamily, CaM kinases share structural and functional homology both in thekinase catalytic domain and in a regulatory region composed of compositeautoinhibitory and CaM binding domains (Hanks et al., Methods Enzymol.,200:38–79 (1991); Hanks et al., Science, 241:42–52 (1988); Haribabu etal., EMBO J., 14:3679–3686 (1995); Knighton et al., Science, 258:130–135(1992); Picciotto et al., I. Adv. Pharmacol., 36:251–275 (1996);Yokokura et al., J. Biol. Chem., 270:23851–23859 (1995)).

Despite these similarities, significant differences exist between CaMkinase family members. For instance, this family includes members withhigh substrate specificity, such as myosin light-chain kinase (MLCK) andphosphorylase kinase, as well as members with broader substratespecificities collectively referred to as the multifunctional CaMkinases, such as CaMKI, CaMKIV, and members of the CaMKII subfamily(Braun et al., Annu. Rev. Physiol., 57: 417–445 (1995); Cawley et al.,J. Biol. Chem., 268:1194–1200 (1993); Herring et al., J. Biol. Chem.,265:1724–1730 (1990); Matthews et al., 1994; Schulman, 1993). Otherproperties that differ among CaM kinase family members include theirsubcellular localization, regulation by autophosphorylation, andregulation by other proteins. In addition, CaM kinases have uniqueamino- and carboxyl-terminal domains that contribute to kinase-specificdifferences in subcellular localization, subunit interactions, and otherprotein-protein interactions. Much of the information availableregarding the multifunctional CaM kinases is derived from studiesconducted in the brain, where they are expressed at high levels.

In light of these findings, it is clear that until the presentinvention, there has remained a need to identify and study the role ofprotein kinases in postnatal development and carcinogenesis, as well asprovide insight into how the decision to proliferate or differentiate ismade in mammary epithelial cells. Moreover, identification ofcancer-linked protein expression product, and the gene encoding same,offers previously unavailable diagnostic and therapeutic solutions tocarcinogenesis.

SUMMARY OF THE INVENTION

The present invention was the product of a systematic study of the roleof protein kinases in mammary gland development and carcinogenesis.Based upon examination of defined stages in postnatal mammarydevelopment and in a panel of mammary epithelial cell lines derived fromdistinct transgenic models of breast cancer, the inventors discovered anovel serine/threonine kinase, Pnck (Pregnancy-Up-regulatedNonubiquitous CaM Kinase). The isolation of Pnck resulted from theexamination of 1450 cDNA clones generated using a RT-PCR-based screeningstrategy, which identified 41 protein kinases, including 33 tyrosinekinases and 8 serine/threonine kinases, 3 of which were novel.

The PNCK kinase has been shown to be highly overexpressed in a subset ofhuman breast cancers compared to benign breast tissue. In addition,expression of the PNCK kinase has been shown to be elevated in humanovarian carcinomas compared to benign tissue and to be positivelycorrelated with tumor grade. In other words, the higher the tumor grade,the higher the expression of the PNCK kinase). Conversely, expression ofthe PNCK kinase has been shown to be decreased in human colon carcinomascompared to benign tissue and to be negatively correlated with tumorgrade. Such a correlation between the genes of the present invention andvarious cancers has not been previously reported, although it is unclearat this point whether the altered expression of the kinase is acoincidental marker of tumor behavior, or whether the altered expressionof the kinase is causally related to the cancer.

The present invention provides the purified cancer-linked proteinkinase, Pnck, and the isolated nucleotide sequence encoding the kinase.

The present invention further provides a method of delivering Pnck to atarget cell, wherein the method comprises delivering to the target cellan effective amount of the kinase, or of the nucleotide sequenceencoding the kinase. In preferred embodiments of the invention theamount of the kinase or the gene encoding the kinase delivered to thepatient is a therapeutically effective amount. In addition, the kinasecan act as a marker of target cell activity.

The present invention also provides a method of delivering an effectiveamount of an inhibitor of the Pnck kinase to block the activation of, ordecrease the activity of, the kinase in the target cell. In particular,the delivered inhibitor comprises an antisense or anti-Pnck molecule. Inat least one embodiment, the kinase is overexpressed in the target cell,as compared with a comparable normal cell of the same type. In thealternative, a method is provided for delivering an effective amount ofa composition to activate or increase the activity of the Pnck or thenucleotide sequence encoding the kinase in the target cell. In at leastone embodiment, the kinase is underexpressed in the target cell, ascompared with a comparable normal cell of the same type.

In addition, the invention provides a method of treating cancer,hyperproliferative disease or oncogene expression in a patient, whereinthe method comprises delivering to a target cell in the patient atherapeutically effective amount of Pnck or of the nucleotide sequenceencoding Pnck. As in the previously described method of delivery, themethod of treatment comprises delivering an effective amount of aninhibitor of the Pnck kinase to block the activation of, or decrease theactivity of, the kinase in the target cell. In particular, the deliveredinhibitor comprises an antisense or anti-Pnck molecule. In at least oneembodiment, the kinase is overexpressed in the target cell, as comparedwith a comparable normal cell of the same type. In the alternative, amethod is provided for treating cancer, hyperproliferative disease oroncogene expression in a patient comprising delivering an effectiveamount of a composition to activate or increase the activity of Pnck orthe nucleotide sequence encoding the kinase in the target cell. In atleast one embodiment, the kinase is underexpressed in the target cell,as compared with a comparable normal cell of the same type.

The present invention further provides a method of diagnosing a cancer,carcinoma, sarcoma, neoplasm, leukemia, lymphoma or hyperproliferativecell disease or oncogene expression in a patient, wherein the methodcomprises detecting the presence of and/or measuring Pnck activity or achange therein, as compared with a comparable normal cell of the sametype. The method effectively detects and/or measures either theoverexpression or under expression of Pnck.

Also provided is a method of rapid screening for a selected compoundthat modulates the activity of Pnck, comprising: (i) quantifying theexpression of the kinase from a target cell; (ii) treating the targetcell by administering thereto the selected compound, wherein all otherconditions are constant with those in the quantifying step; (iii)quantifying the expression of the kinase from the treated target cell;and (iv) comparing the two quantification measurements to determine themodulation of kinase activity achieved by treatment with the selectedcompound. The method is applicable to screening for either the presenceof kinase, or an underexpression or a measurable decrease in kinaseactivity, or an overexpression or a measurable increase in kinaseactivity. It further extends to transformation of the target cell.

Further provided is a method of using Pnck or the nucleotide sequenceencoding Pnck as a prognostic tool in a patient to detect the presenceof, and/or measure the activity or change of activity of the kinase, asa molecular marker in the patient to predict the behavior of a tumor,cancer, carcinoma, sarcoma, neoplasm, leukemia, lymphoma orhyperproliferative cell disease or oncogene expression in the patient,and applying that detection to predict the appropriate therapy for thepatient.

It is particularly preferred that the target cell of the methods of thepresent invention is human, and that the patient is human.

In addition, the present invention provides a recombinant cellcomprising Pnck or PNCK, or a vector or recombinant cell comprisingsame. Also provided is an antibody specific for the Pnck or PNCK, andhomologues, analogs, derivatives or fragments thereof having Pnckactivity; as well as an isolated nucleic acid sequence comprising asequence complementary to all or part of the Pnck or PNCK, and tomutants, derivatives, homologues or fragments thereof encoding a cellhaving Pnck activity. A preferred complementary sequence comprisesantisense activity at a level sufficient to regulate, control, ormodulate Pnck activity in a target cell expressing the kinase. Alsoincluded in the present invention is a transgenic cell and/or atransgenic animal comprising Pnck or PNCK, or the nucleic acid encodingsame.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description, examples and figures whichfollow, and in part will become apparent to those skilled in the art onexamination of the following, or may be learned by practice of theinvention.

DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. For the purpose of illustrating the invention,there are shown in the drawings, certain embodiment(s), which arepresently preferred. It should be understood, however, that theinvention is not limited to the precise arrangements andinstrumentalities shown.

FIG. 1 depicts a composite nucleic acid sequence (SEQID No. 1) andconceptual translation (SEQID No. 2) of full-length Pnck cDNA.Nucleotide coordinates are shown on the left. Amino acid coordinates areshown in boldface type on the right. A shaded box indicates the kinasecatalytic domain, and a hatched box denotes the putative regulatoryregion. The in-frame upstream termination codons in the 5′-UTR and theputative polyadenylation sequence in the 3′-UTR are underlined by thinand thick lines, respectively. The putative initiation codon is boxed,and an asterisk denotes the stop codon. Arrows underline the regionscorresponding to the degenerate oligonucleotides used to clone Bstk3initially.

FIGS. 2A and 2B shows the expression and coding potential of Pnck. FIG.2A depicts a Northern hybridization analysis of poly(A)⁺ RNA isolatedfrom adult murine brain hybridized with a 3′-UTR probe specific forPnck. The relative migration of RNA size markers is indicated. FIG. 2Bdepicts in vitro transcription/translation reactions performed using³⁵S-labeled methionine-labeled reticulocyte lysates with a full-lengthPnck cDNA clone (V1, U7, or Q3), or a cDNA plasmid encoding an unrelatedkinase (−) as a negative control. IVT reactions were resolved on a 10%SDS-PAGE gel. The relative migration of molecular weight markers isindicated.

FIG. 3 depicts a segregation analysis of Pnck within the central regionof the mouse X chromosome. FIG. 3A graphically shows that thesegregation patterns of Pnck and flanking genes in the loci are shown atthe top of the figure. Each column of FIG. 3A represents the chromosomeidentified in the backcross progeny that was inherited from the(C57BL/6JX M. spretus) F₁ parent. The shaded boxes in FIG. 3A representthe presence of a C57BL/6J allele, and white boxes represent thepresence of a M. spretus allele. The number of offspring inheriting eachtype of chromosome is listed at the bottom of each column in FIG. 3A. Apartial X chromosome linkage map showing the location of Pnck inrelation to linked genes is shown in FIG. 3B. Recombination distancesbetween loci in centimorgans are shown to the left of the chromosome,and the positions of loci in human chromosomes are shown to the right.(References for the human map positions of cited loci from GDB (GenomeData Base)).

FIGS. 4A and 4B depict the expression of Pnck during murineembryogenesis. FIG. 4A depicts a Northern hybridization analysis of 3 mgof poly(A)⁺RNA isolated from day E6.5, E13.5, and E18.5 embryoshybridized with a ³²P-labeled DNA probe specific for the 3′-UTR of Pnck.FIG. 4B depicts in situ hybridization analysis of Pnck mRNA expressionin the murine embryo. Sections of embryos at day 14.5 of gestation werehybridized with a ³⁵S-labeled Pnck anti-sense RNA probe. Nosignal-over-background was detected in serial sections hybridized with asense Pnck probe. bo=bone; bt=basal telen-cephalon; fv=fourth ventricle;li=liver; lu=lung; lv=lateral ventricle; st=stomach; tg=trigeminalganglion; wf=whisker hair follicle. Magnification: 10×. Exposure timewas 6 weeks.

FIGS. 5A–5M depict expression of Pnck in adult tissues. FIG. 5A depictsRNase protection analysis of Pnck mRNA expression in indicated tissuesof the adult mouse using antisense RNA probes specific for Pnck, as wellas for β-actin as an internal control. tRNA was used as a negativecontrol for nonspecific hybridization. FIGS. 5B–5M depict spatiallocalization of Pnck expression in tissues of the adult mouse. FIGS. 5B,5D, 5F, 5H, 5J, 5L depict dark-field and FIGS. 5C, 5E, 5G, 5I, 5K, 5Mdepict bright field photomicrographs of in situ hybridization analysisperformed on sections of brain (FIGS. 5B, 5C, 5F, 5G, 5J, 5K), testis(FIGS. 5D, 5E), ovary (FIGS. 5H, 5I), and prostate (FIGS. 5L, 5M),hybridized with an ³⁵S-labeled Pnck antisense probe. Nosignal-over-background was detected in serial sections hybridized with acorresponding sense Pnck probe. Arrows and arrowheads indicate Pnckexpressing and Pnck nonexpressing cells, respectively. bm, basementmembrane; CA1 and CA3, regions of the hippocampus; co=cortex; d=duct;dg=dentate gyrus; fo=follicle; se=seminiferous tubule; sp=spermatids;st=stroma. Magnifications: 10× for FIGS. 5B and 5C; 300× for FIGS.5D–5M. Exposure times were 6–7 weeks.

FIGS. 6A and 6B depict the temporal regulation of Pnck expression duringmurine mammary gland development. FIG. 6A depicts a RNase protectionanalysis of Pnck mRNA expression during postnatal murine mammary glanddevelopment. Each indicated developmental time point represents isolatedmammary gland total RNA hybridized to ³²P-labeled antisense riboprobesspecific for the 3′ untranslated region of Pnck, or for β-actin. FIG. 6Bdepicts phosphorimager quantification of RNase protection analysis shownin FIG. 6A. Expression levels are shown relative to matched adult virginanimals.

FIGS. 7A–7J depict spatial regulation of Pnck expression in the mammarygland during development. Bright-field panels (FIGS. 7A, 7C, 7E, 7G, 7I)and dark-field panels (FIGS. 7B, 7D, 7F, 7H, 7J), respectively, depictphotomicrographs of mammary gland sections hybridized in situ with³⁵S-labeled Pnck-specific antisense riboprobe. No signal-over-backgroundwas detected in serial sections hybridized with the corresponding sensePnck probe. Exposure times were identical (7 weeks) for all dark-fieldphotomicrographs to illustrate changes in Pnck expression duringpregnancy and lactation. Arrows point to Pnck-expressing epithelialcells. al=alveoli; d=duct; lo=alveolar lobule; st=adipose stroma;teb=terminal end bud. Magnification: 300×.

FIGS. 8A and 8B depicts proliferation-dependent expression of Pnck. FIG.8A depicts an RNase protection analysis of Pnck expression in activelygrowing versus confluent cells. ³²P-labeled antisense riboprobesspecific for Pnck or Gapdh were hybridized with total RNA isolated fromthe indicated cell lines, while either actively growing (Act) or 3 daysafter confluence (Con). FIG. 8B depicts an RNase protection analysis ofPnck expression in serum-starved 16 MB9a cells at the indicated timesafter re-feeding.

FIG. 9 depicts Pnck expression in nontransformed and transformed murinemammary epithelial cell lines. Transformed cell lines were derived frommammary adenocarcinomas arising in mouse mammary tumor virus (MMTV)transgenic mice expressing the int-2/Fgf3, c-myc, neu, or H-rasoncogenes in the mammary gland. RNase protection analysis was performedon poly(A)⁺ RNA isolated from actively growing murine cell lineshybridized with a ³²P-labeled antisense riboprobe specific for the 3′untranslated region of Pnck (top panel). Northern analysis was performedon poly(A)⁺ RNA using ³²P-labeled cDNA probes specific for c-myc (middlepanel) or cyclin D3 (bottom panel). The poly(A)⁺ RNA beneath the 28SrRNA band is shown as a loading control. Cell lines are: NIH-3T3fibroblast, nontransformed (Non-Tx): Lane 1, NMuMG, Lane 2, HC11, andLane 3, CL-S1. MMTV-int-2/Fgf3: Lane 4, HBI2; and Lane 5, 1128.MMTV-c-myc: Lane 6, 8MA1a; Lane 7, MBp6; Lane 8, M1011; Lane9, M158; andLane 10, 16 MB9a. MMTV-neu: Lane 11, SMF; Lane 12, NaF; Lane 13, NF639;Lane 14, NF11005; and Lane 15, NK-2. MMTV-H-ras: Lane 16, AC816; Lane17, AC711; and Lane 18, AC236.

FIG. 10 depicts PNCK expression in a subset of human breast tumor celllines. RNase protection analysis of was performed using actively growinghuman breast tumor cell lines hybridized with a ³²P-labeled antisenseriboprobe specific for PNCK, or for β-actin. Cell lines are: Lane 1,184B5; Lane 2, 2 MT-2; Lane 3, BT-20; Lane 4, BT-474; Lane 5, BT-549;Lane 6, HBL-100; Lane 7, MDA-MB-157; Lane 8, MDA-MB-231; Lane 9,MDA-MB-361; Lane 10, MDA-MB-435; Lane 11, MDA-MB-436; Lane 12MDA-MB-453; Lane 13, MDA-MB-468; Lane 14, SK-BR-3; Lane 15, ZR-75-1;Lane 16, MCF-10; Lane 17, MCF-10A; and Lane 18, Hs 578T

FIG. 11A–11C depict the overexpression of PNCK in a subset of humanprimary breast tumors, as seen in 12 benign breast tissue samples and 23primary breast tumors. In FIG. 11A, RNase protection analysis wasperformed using total RNA hybridized with a ³²P-labeled anti senseriboprobe specific for PNCK or β-actin, as indicated. Northernhybridization analysis was performed on the same RNA samples using totalRNA hybridized with a ³²P-labeled cDNA probe specific for cytokeratin 18(CK18). The 28S rRNA band is shown as a control for equal RNA loading.Expression levels were quantified by phosphorimager analysis, and PNCKexpression levels normalized to CK18 are shown for each sample. In FIG.11B, PNCK expression levels in breast tumors, normalized either toβ-actin or to CK18, as indicated, are compared with benign tissue,wherein normalized PNCK expression levels in benign tissues was setequal to 1.0. The mean of each distribution is shown. Bars, SE. P=0.01for PNCK/β-actin expression in tumors compared with benign tissue. ‡,P=0.039 for PNCK/CK18 expression in tumors compared with benign tissue.FIG. 11C shows a histogram of individual PNCK expression levelsnormalized to CK18 for the samples shown in FIG. 11A. PNCK expressionfor each sample was normalized to CK18 expression, and the averageexpression in benign samples was set equal to 1.0. Displayed valuesrepresent fold changes relative to the mean PNCK/CK18 expression levelobserved for benign breast tissue. Bin sizes are 0.5 unit. Mode is samefor both tumor and benign samples.

DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides the first data implicating a CaM kinasein mammary development or carcinogenesis. To better understand therelationship between development and carcinogenesis in the breast, ascreen was designed to identify protein kinases that are expressed inthe murine mammary gland during development and in mammary tumor celllines Chodosh et al., Dev. Biol., 219:259–276 (2000); Gardner et al.,Genomics, 63:46–59 (2000); Gardner et al., Genomics, 63:279–288 (2000);Stairs et al., Hum. Mol. Genet., 7:2157–2166 (1998)). After kinases wereclustered on the basis of similarities in their temporal expressionprofiles during mammary development, multiple distinct patterns ofexpression were observed. Analysis of these patterns revealed an orderedset of expression profiles in which successive waves of kinaseexpression occur during development. This resulted in the identificationof a novel serine/threonine kinase from the mammary glands of miceundergoing early postlactational involution, specifically aPregnancy-Up-regulated, Nonubiquitous CaM Kinase (PNCK), so named toreflect its temporally and spatially regulated pattern of expression inthe mammary gland.

The invention provides the Pnck gene, which has been cloned and fullysequenced as described in the Examples below, and the full length codingsequence derived from cDNA is set forth in FIG. 1 and SEQID No:1.Sequence data have been deposited with the EMBL/GenBank® Data Librariesunder Accession No. AF181984.

“Homologous” as used herein, refers to the subunit sequence similaritybetween two polymeric molecules, e.g., between two nucleic acidmolecules, e.g., two DNA molecules or two RNA molecules, or between twopolypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions, e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two compound sequences are homologous then the twosequences are 50% homologous, if 90% of the positions, e.g., 9 of 10,are matched or homologous, the two sequences share 90% homology. By wayof example, the DNA sequences 3′ATTGCC5′ and 3′TATGCG5′ share 50%homology.

In the Examples that follow, two homologous genes are examined, andwhile not intended to be limited to the exemplified species, standardnomenclature is used. The murine gene is referred to as Pnck, whereas asthe human homologue of the same gene is referred to as PNCK. Thenucleotide sequence for human PNCK is set forth as SEQID NO:7, and itscorresponding protein expression product as SEQID NO:8. Thus, theinvention should be construed to include all Pnck kinase genes that meetthe description herein provided, including the human homologue PNCK, asherein described.

The gene encoding Pnck kinase may be isolated as described herein, or byother methods known to those skilled in the art in light of the presentdisclosure. Alternatively, since, according to the present invention,the gene encoding Pnck has been identified, isolated and characterized,any other Pnck gene which encodes the unique protein kinase describedherein may be isolated using recombinant DNA technology, wherein probesderived from Pnck are generated which comprise conserved nucleotidesequences in kinase genes. These probes may be used to identifyadditional protein kinase genes in genomic DNA libraries obtained fromother host strain using the polymerase chain reaction (PCR) or otherrecombinant DNA methodologies.

An “isolated nucleic acid,” as used herein, refers to a nucleic acidsequence, segment, or fragment which has been separated from thesequences which flank it in a naturally occurring state, e.g., a DNAfragment which has been removed from the sequences which are normallyadjacent to the fragment, e.g., the sequences adjacent to the fragmentin a genome in which it naturally occurs. The term also applies tonucleic acids which have been substantially purified from othercomponents which naturally accompany the nucleic acid, e.g., RNA or DNAor proteins which naturally accompany it in the cell.

Further provided in the present invention is the isolated polypeptideprotein kinase product of the Pnck gene and its biological equivalents,which are useful in the methods of this invention. Preferably, the aminoacid sequence of the isolated protein kinase is about 70% homologous,more preferably about 80% homologous, even more preferably about 90%homologous and most preferably about 95% homologous to the amino acidsequence Pnck, or its human homologue, PNCK.

The expression product of the Pnck gene, encodes a 38-kDa protein kinaseset forth in FIG. 1 and SEQID No:2. The coding sequence for Pnck isdivided into a 14-amino-acid unique amino-terminal segment, a 256amino-acid kinase catalytic domain, a 41-amino-acid regulatory domain,and a 32-amino-acid unique carboxyl-terminal region. The Pnck kinasecatalytic domain contains all of the amino acid motifs conserved amongserine/threonine kinases. The catalytic domain of Pnck shares 45–70%identity with members of the Ca²⁺/calmodulin-dependent (CaM) family ofprotein serine/threonine kinases.

While this work was in progress, a rat CaM was identified and shown tobe expressed as two isoforms, tentatively named CaMKIβ1 and CaMKIβ2(Naito et al., J. Biol. Chem., 272: 32704–32708 (1997)). Althoughsimilar to the clones isolated for Pnck, CaMKIβ1 and CaMKIβ2 differ intheir 5′-UTR regions and are homologous to Pnck clones V1 and U7,respectively. However, unlike the full-length clones isolated for Pnck,CaMKIβ1 contains a unique carboxyl-terminal coding region that appearsto result from an alternative splicing event. The 3′ end of each CaMmember molecule is unique, differentiating and distinguishing themembers from one another. Consequently, although regions of theidentified isoforms are homologous to regions in Pnck, the identifiedrat genes and Pnck are different, presumably having differentcharacteristics and functions.

Northern hybridization analysis using a probe encompassing portions ofthe highly conserved kinase domain and regulatory region of CaMKIβisoforms detected a 1.8-kb band exclusively in brain, whereas a 4.0-kbband was detected in all other tissues. By way of contrast, the mRNAencoding Pnck is 1.5-kb in length in all tissues examined. Reversetranscriptase (RT)-PCR analysis detected approximately equal levels ofCaMKIβ1 in all tissues examined in the rat. This included tissues inwhich Pnck in the mouse is expressed at only low or undetectable levels,as determined by RNase protection analysis using a probe specific forthe 3′-UTR of Pnck. Insofar as the tissue-specific expression pattern ofPnck has been confirmed by i1i situ hybridization analysis, and giventhe numerous differences between Pnck and its expression product, andthe gene reported as CaMKIβ and its expression product, the two are notthe same, despite regions of homology. One of ordinary skill in the artwould not have been led to the discovery of the full-length Pnck, norwould the unique spatial and temporal characteristics associated withPnck expression have been suggested by the Nairn et al. report.

The Pnck gene locus has been mapped in mouse to within 2.2 cM of Il1rakin the central region of the X chromosome, in a region of conservedsynteny with human chromosome Xq28, strongly suggesting that the humanhomologue of Pnck will also map to Xq28. Chromosome Xq28 is one of themost densely mapped regions of the human chromosome, frequentlyassociated with mental retardation syndromes (Lubs et al., Am. J. Med.Genet., 83:237–247 (1999)).

Pnck can be purified from natural sources or produced recombinantlyusing the expression vectors described above in a host-vector system.The proteins also can be produced using the sequence provided in FIG. 1,and by methods well known to those of skill in the art. The isolatedpreparation of Pnck kinase encoded by Pnck may be obtained by cloningand expressing the Pnck gene, and isolating the Pnck protein soexpressed, using available technology in the art, and as describedherein. The kinase may be purified by following known procedures forprotein purification, wherein an immunological, enzymatic or other assayis used to monitor purification at each stage in the procedure.

A “biological equivalent” is intended to mean any fragment of thenucleic acid or protein, or a mimetic (protein and non-protein mimetic)also having the ability to inhibit Pnck kinase activity using the assaysystems described and exemplified herein. For example, purified Pnckpolypeptide can be contacted with a suitable cell, as described above,and under such conditions that its kinase activity is inhibited, or inthe alternative enhanced. By “inhibited,” is meant a change in kinaseactivity that is measurably less than the activity exhibited beforecontact with the subject cell; by “enhances,” is meant a change inkinase activity that is measurably greater than the activity exhibitedbefore contact with the subject cell.

The protein is used in substantially pure form. As used herein, the term“substantially pure,” or “isolated preparation of a polypeptide” ismeant that the protein is substantially free of other biochemicalmoieties with which it is normally associated in nature. Typically, acompound is isolated when at least 25%, more preferably at least 50%,more preferably at least 60%, more preferably at least 75%, morepreferably at least 90%, and most preferably at least 99% of the totalmaterial (by volume, by wet or dry weight, or by mole percent or molefraction) in a sample is the compound of interest. Purity can bemeasured by any appropriate method, e.g., in the case of polypeptides bycolumn chromatography, gel electrophoresis or HPLC analysis.

The present invention also provides for analogs of proteins or peptidesencoded by Pnck or its human homologue, PNCK. Analogs can differ fromnaturally occurring proteins or peptides by conservative amino acidsequence differences or by modifications which do not affect sequence,or by both. It is understood that limited modifications can be made tothe primary sequence of the Pnck sequence as shown in FIG. 1 and used inthis invention without destroying its biological function, and that onlya portion of the entire primary structure may be required in order toeffect biological activity. It is further understood that minormodifications of the primary amino acid sequence may result in proteins,which have substantially equivalent or enhanced function as compared tothe molecule within the vector. These modifications may be deliberate,e.g., through site-directed mutagenesis, or may be accidental, e.g.,through mutation in hosts. All of these modifications are included inthe present invention, as long as the Pnck kinase activity is retainedessentially as in its native form.

For example, conservative amino acid changes may be made, which althoughthey alter the primary sequence of the protein or peptide, do notnormally alter its function. Conservative amino acid substitutionstypically include substitutions within the following groups: glycine andalanine; valine, isoleucine, and leucine; aspartic acid and glutamicacid; asparagine and glutamine; serine and threonine; lysine andarginine; or phenylalanine and tyrosine.

Modifications (which do not normally alter primary sequence) include invivo, or in vitro chemical derivatization of polypeptides, e.g.,acetylation, or carboxylation. Also included are modifications ofglycosylation, e.g., those made by modifying the glycosylation patternsof a polypeptide during its synthesis and processing or in furtherprocessing steps, e.g., by exposing the polypeptide to enzymes whichaffect glycosylation, e.g., mammalian glycosylating or deglycosylatingenzymes. Also embraced are sequences that have phosphorylated amino acidresidues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.Also included are polypeptides that have been modified using ordinarymolecular biological techniques so as to improve their resistance toproteolytic degradation or to optimize solubility properties. Analogs ofsuch polypeptides include those containing residues other thannaturally-occurring L-amino acids, e.g., D-amino acids or non-naturallyoccurring synthetic amino acids. The peptides of the invention are notlimited to products of any of the specific exemplary processes listedherein.

In addition to substantially full-length polypeptides, the presentinvention provides for enzymatically active fragments of thepolypeptides. A Pnck-specific polypeptide is “enzymatically active” ifit is characterized in substantially the same manner as the naturallyencoded protein in the assays described below.

As used herein, the term “fragment,” as applied to a polypeptide, willordinarily be at least about 20 contiguous amino acids, typically atleast about 50 contiguous amino acids, more typically at least about 70continuous amino acids, usually at least about 100 contiguous aminoacids, more preferably at least about 150 continuous amino acids inlength.

Pnck is spatially and temporally regulated during murine mammarydevelopment with highest levels of expression observed late in pregnancyas alveolar epithelial cells exit the cell cycle and undergo terminaldifferentiation. Pnck expression is developmentally up-regulated andtissue-specific during intermediate and late stages of murine fetaldevelopment with highest levels of expression in developing brain, bone,and gastrointestinal tract. By comparison, in adult mice, highest levelsof Pnck expression are found in the brain, particularly in thehippocampus and dentate gyrus, and in the uterus, ovary, and testis.

Potentially related to this temporal pattern of expression, Pnck isup-regulated in serum-starved and confluent mammary epithelial cells,and down-regulated as serum-starved cells are stimulated to reenter thecell cycle. Thus, the up-regulation of Pnck expression in the mammarygland late in pregnancy may be related to the decreased proliferation ofmammary epithelial cells during this stage of development. Moreover,Pnck expression in the mammary gland is restricted to a subset ofepithelial cells during development indicating an involvement of thiskinase in a variety of developmental processes.

Interestingly, Pnck expression in adult animals is both tissue-specificand markedly heterogeneous within several tissues, with expressing cellsfound adjacent to non-expressing cells. Pnck expression is alsorestricted to particular compartments, and is further found to berestricted to subsets of cells within those compartments. For example,Pnck expression is limited to particular epithelial or stromalcompartments, and within these compartments, Pnck expression is furtherrestricted to a subset of cells.

Moreover, Pnck is expressed in an oncogene-associated manner in celllines derived from murine mammary tumors with defined initiating events.Similarly, expression of the human homologue of Pnck is restricted to asubset of human breast tumor cell lines and is highly overexpressed in asubset of primary human breast cancers, when compared with benign breasttissue. In aggregate, these data are consistent with the understandingthat PNCK expression is restricted to a subset of ductal carcinomas inhumans, and suggest a role for PNCK, or a cell type that expresses thePNCK gene, in mammary carcinogenesis.

Both the up-regulation of Pnck observed in confluent cells and thedown-regulation of Pnck observed as serum-starved cells reenter the cellcycle are consistent with Pnck expression patterns in the mammary glandduring late pregnancy. Although the up-regulation of Pnck observedduring late pregnancy may be an effect of decreased epithelialproliferation, it also indicates a direct involvement by Pnckup-regulation in inhibiting cellular proliferation or contributing tothe exit of epithelial cells from the cell cycle prior to their terminaldifferentiation. Thus, Pnck appears to be involved in cell cycleregulation.

Certain catalytic and regulatory domains are conserved among all membersof the CaM-dependent family of protein kinases. Pnck is most closelyrelated to the multifunctional CaM kinases, CaMKI, CaMKIV, and membersof the CaMKII subfamily. Both calmodulin and CaM-dependent kinases havebeen reported previously to be involved in cell cycle progression. Thepresent data demonstrate that Pnck expression in vitro is inverselycorrelated with cellular proliferation. Specifically, decreasing theproliferative status of mammary epithelial cells in vitro resulted inincreased Pnck expression.

Ca²⁺ is a key intracellular signaling molecule that exerts some of itseffects by binding to calmodulin and activating CaM kinases, andcalmodulin has been implicated in development. However, developmentalroles for multifunctional CaM kinases, including CaMKI, have not beendefined.

CaMKI is a monomeric kinase that is expressed in multiple tissues and isreported to phosphorylate several substrates including synapsin, thecystic fibrosis transmembrane conductance regulator, and transcriptionfactors, such as the cyclic AMP response element-binding protein, CREBand ATF-1 (Lukas et al., 1998; Nairn et al., 1994; Nastluk et al., I.Adv. Pharmacol., 36:251–275 (1996); Sheng et al., 1991). CaMKIV islocated in the nucleus, and has been proposed to mediate CaM-inducedchanges in gene expression (Jensen et al., Proc. Natl. Acad. Sci. USA,88:2850–2853 (1991A); Sun et al., J. Biol. Chem., 271:3066–3073 (1996)).

In contrast to CaMKI and IV, which function as monomers, CaMKII forms300- to 600-kDa multimers composed of different combinations of a, b, g,and d subunits (Schulman, 1993). While the a and b subunits areexpressed predominantly in brain, the g and d CaMKII subunits areexpressed ubiquitously (Hanley et al., Science, 237:293–297 (1987); Linet al., Proc. Natl. Acad. Sci. USA, 84:5962–5966 (1987); Tobimatsu etal., J. Biol. Chem., 264:17907–17912 (1989); Tobimatsu et al., J. Biol.Chem., 263:16082–16086 (1988)).

Functional analysis of CaM kinase mutants as well as crystal structureinformation has been used to define amino acids involved in theregulation of this family of molecules (Goldberg et al., Cell,84:875–887 (1996); Haribabu et al., 1995); Yokokura et al., 1995).Carboxyl-terminal to their catalytic domain, CaM kinases possess aregulatory region that is composed of an autoinhibitory domain and aCaM-binding domain. In contrast to other CaM kinases, the activities ofCaMKI and CaMKIV are dependent upon phosphorylation by a CaM-dependentkinase, CaMKK (Haribabu et al., 1995; Tokumitsu et al., 1994, 1995).

The homology between Pnck and CaMKI raises the issue of whether Pnckshould be classified as a CaMKI family member. Currently, the onlywidely recognized CaM kinase subfamily is that of CaMKII. Primary aminoacid sequences of CaMKII subfamily members are typically greater than90% identical in the catalytic and regulatory domains and actuallyfunction together in a multiprotein complex. In contrast, while the 70%amino acid identity in the catalytic domain between Pnck and CaMKI isgreater than that between Pnck and other CaM kinases, the similaritybetween Pnck and CaMKI is significantly less than the approximately 90%identity observed between CaMKII family members. Moreover, there iscurrently no evidence to suggest that CaMKI family members function assub-units in a manner analogous to CaMKII subfamily members. As such,while the primary amino acid sequence of Pnck is more similar to that ofCaMKI than to other CaM molecules, and while Pnck may have functionsunique to this family of molecules, it is unclear at present that thiskinase should be classified as a CaMKI family member.

While expression of CaMKI, CaMKIV and isoforms of CaMKII has beenreported in tissues other than the brain, a physiological role for theseenzymes in other tissues has not been described. Interestingly, to date,the only biological role described for any of the multi-functional CaMkinases is that of CaMKII in learning and memory.

Similar to the CaM multifunctional kinases, Pnck is expressed in avariety of tissues other than the brain. Moreover, in most tissuesexamined, Pnck is expressed in a spatially heterogeneous manner withexpression restricted to a subset of cells. The observation that Pnckexpression is developmentally regulated and spatially restricted todistinct compartments of the ovary, testis, prostate, and brain suggeststhat Pnck may play a biological role in these tissues. As such, theelucidation of signaling pathways in which Pnck is involved may shedlight on the broader physiological role played by CaM kinases.

Functionally, the temporal and spatial regulation of Pnck has beencharacterized in various murine and human tissues, as summarized inTable 1.

TABLE 1 Pnck Expression Expression Expressed in terminallydifferentiating (non-proliferating cells) in vivo; Up-regulated innon-proliferating cells in vitro. Breast Cancer in Not expressed in celllines from tumors induced by the Transgenic Mice Neu/ErbB2/Her2 and Rasoncogenes; Overexpressed in cell lines from tumors induced by the c-Mycor Int-2 oncogenes. Expression in Expression is highly heterogeneous incell lines from a Human Cancer wide variety of tumor types; expressed athigh levels Cell Lines (or at undetectable levels) in a subset ofbreast, colon, ovarian, prostate, lung, CNS, cervical and renal cancercell lines. Human Breast Overexpressed in a subset of primary breastcancers Cancer compared to benign tissue. Human Colon Under expressed incolon cancers compared to Cancer benign tissue. Human OvarianOverexpressed in ovarian cancers compared to Cancer benign tissues.Other Human Overexpressed in a subset of endometrial cancers Cancerscompared to benign tissue. Highly expressed in a subset of carcinoidtumors.

The invention further includes a method of identifying a therapeuticcompound having activity to affect Pnck by screening a test compound forits ability to modulate the expression or activity of Pnck. Suchcompounds may include antibiotics. In addition, these kinases will beuseful diagnostically, as markers to assess a patient's illness, and/orprognostically, to determine how aggressively, or with what agent adiagnosed case of cancer should be treated.

Methods of the invention can be practiced in vitro, ex vivo or in vivo.When the method is practiced in vitro, the expression vector, protein orpolypeptide can be added to the cells in culture or added to apharmaceutically acceptable carrier as defined below. In addition, theexpression vector or Pnck DNA can be inserted into the target cell usingwell known techniques, such as transfection, electroporation ormicroinjection. By “target cell” is meant any cell that is the focus ofexamination, delivery, therapy, modulation or the like by, or as aresult of, activation, inactivation, expression or changed expression ofPnck or the nucleotide sequence encoding same, or any cell that effectssuch modulation, activation, inactivation or the like in the kinase orgene encoding it.

Compounds which are identified using the methods of the invention arecandidate therapeutic compounds for treatment of disease states orcarcinomas in patients caused by or associated with Pnck or by a celltype related to the activation of Pnck, such as an epithelial cell typeas yet unidentified, which activates or is activated by a cancerouscondition in the subject, particularly in a human patient. By “patient”is meant any human or animal subject in need of treatment and/or to whomthe compositions or methods of the present invention are applied. It ispreferred that the patient of the present invention is a mammal, morepreferred that it is a veterinary animal, most preferred that it is ahuman.

The use of the compositions and methods in vitro provides a powerfulbioassay for screening for drugs that are agonists or antagonists ofPnck function in these cells. Thus, one can screen for drugs havingsimilar or enhanced ability to prevent or inhibit Pnck kinase activity.It also is useful to assay for drugs having the ability to inhibitcarcinogenesis, particularly in the breast. The in vitro method furtherprovides an assay to determine if the method of this invention is usefulto treat a subject's pathological condition or disease that has beenlinked to enhanced Pnck expression, to the developmental stagesassociated with up-regulation of Pnck, or to a cancerous condition,particularly in the breast or other tissues in which Pnck is highlyexpressed.

Generally the term “activity,” as used herein, is intended to relate toPnck kinase activity, and an “effective amount” of a compound withregard to Pnck kinase activity means a compound that modulates (inhibitsor enhances) that Pnck activity. However, the term “activity” as usedherein with regard to a compound, also means the capability of thatcompound, that in some way affects Pnck kinase activity, to also destroyor inhibit the uncontrolled growth of cells, particularly cancerouscells, particularly in a tumor, or which is capable of inhibiting thepathogenesis, i.e., the disease-causing capacity, of such cells.Similarly, an “effective amount” of such a compound is that amount ofthe compound that is sufficient to destroy or inhibit the uncontrolledgrowth of cells, particularly cancerous cells, particularly in a tumor,or which is capable of inhibiting the pathogenesis, i.e., thedisease-causing capacity, of such cells. In the alternative, in the caseof an enhancing effect, and “effective amount” is that amount of thecompound that is sufficient to enhance or increase a desired effect ascompared with a corresponding normal cell, or a benign cell.

When the assay methods of the present invention are practiced in vivo ina human patient, it is unnecessary to provide the inducing agent sinceit is provided by the patient's immune system. However, when practicedin an experimental animal model, it may be necessary to provide aneffective amount of the inducing agent in a pharmaceutically acceptablecarrier prior to administration of the Pnck product, to induce Pnckkinase activity. When the method is practiced in vivo, the carryingvector, Pnck polypeptide, polypeptide equivalent, or Pnck expressionvector (as described below) can be added to a pharmaceuticallyacceptable carrier and systemically administered to the subject, such asa human patient or an animal, e.g., mouse, guinea pig, simian, rabbit orrat. Alternatively, antisense Pnck nucleic acid or a Pnck inhibitor orsuspected Pnck inhibitor is administered. Also, it can be directlyinfused into the cell by microinjection. A fusion protein also can beconstructed comprising the Pnck.

Acceptable “pharmaceutical carriers” are well known to those of skill inthe art and can include, but are not limited to any of the standardpharmaceutical carriers, such as phosphate buffered saline, water andemulsions, such as oil/water emulsions and various types of wettingagents.

The assay method can also be practiced ex vivo. Generally, a sample ofcells, such as those in the mammary gland, blood or other relevanttissue, can be removed from a subject or animal using methods well knownto those of skill in the art. An effective amount of antisense Pncknucleic acid or a Pnck inhibitor or suspected Pnck inhibitor is added tothe cells and the cells are cultured under conditions that favorinternalization of the nucleic acid by the cells. The transformed cellsare then returned or reintroduced to the same subject or animal(autologous) or one of the same species (allogeneic) in an effectiveamount and in combination with appropriate pharmaceutical compositionsand carriers.

As used herein, the term “administering” for in vivo and ex vivopurposes means providing the subject with an effective amount of thenucleic acid molecule or polypeptide effective to prevent or inhibitPnck kinase activity in the target cell.

In each of the assays described, control experiments may include the useof mutant strains or cells types that do not encode Pnck. Such strainsare generated by disruption of the Pnck gene, generally in vitro,followed by recombination of the disrupted gene into the genome of hostcell using technology which is available in the art of recombinant DNAtechnology as applied to the generation of such mutants in light of thepresent disclosure. The host may include transgenic hosts.

In one aspect of the assay method of the invention, a compound isassessed for therapeutic activity by examining the effect of thecompound on Pnck kinase activity. In this instance, the test compound isadded to an assay mixture designed to measure protein kinase activity.The assay mixture may comprise a mixture of cells that express Pnck, abuffer solution suitable for optimal activity of the kinase, and thetest compound. Controls may include the assay mixture without the testcompound and the assay mixture having the test compound. The mixture isincubated for a selected length of time and temperature under conditionssuitable for expression of the Pnck kinase as described herein,whereupon the reaction is stopped and the presence or absence of thekinase, or its overexpression is assessed, also as described herein.

Compounds that modulate the Pnck kinase activity, either by enhancing orinhibiting the activity, are easily identified in the assay by assessingthe production of the expression product by the methods exemplified inthe presence or absence of the test compound. A lower level, or minimalamounts of Pnck in the presence of the test compound compared with theabsence of the test compound in the assay mixture is an indication thatthe test compound inhibits the selected kinase activity. Similarly, anincreased, or significantly increased level, or higher amounts of Pnckin the presence of the test compound compared with the absence of thetest compound in the assay mixture is an indication that the testcompound enhances or increases the selected kinase activity.

The method of the invention is not limited by the type of test compoundused in the assay. The test compound is thus a synthetic ornaturally-occurring molecule, which may comprise a peptide orpeptide-like molecule, or it is any other molecule, either small orlarge, which is suitable for testing in the assay. In anotherembodiment, the test compound is an antibody or antisense moleculedirected against Pnck kinase, or its human homologue, or otherhomologues thereof, or even directed against active fragments of Pnckkinase molecules.

Compounds that inhibit Pnck kinase activity in vitro are then tested foractivity directed against PNCK kinase in vivo in humans. Essentially,the compound is administered to the human by any one of the routesdescribed herein, and the effect of the compound is assessed by clinicaland symptomatic evaluation. Such assessment is well known to thepractitioner in the field of developmental biology or those studying theeffect of cancer drugs. Compounds may also be assessed in an in vivoanimal model, as herein described.

Precise formulations and dosages will depend on the nature of the testcompound and may be determined using standard techniques, by apharmacologist of ordinary skill in the art.

The compound may also be assessed in non-transgenic animals to determinewhether it acts through inhibition of Pnck kinase activity in vivo, orwhether it acts via another mechanism. To test this effect of the testcompound on activity, the procedures described above are followed usingnon-transgenic animals instead of transgenic animals.

This invention also provides vector and protein compositions useful forthe preparation of medicaments which can be used for preventing orinhibiting Pnck kinase activity, maintaining cellular function andviability in a suitable cell, or for the treatment of a diseasecharacterized by the unwanted death of target cells or uncontrolled cellamplification, particularly as in a cancer.

The nucleic acid can be duplicated using a host-vector system andtraditional cloning techniques with appropriate replication vectors. A“host-vector system” refers to host cells which have been transfectedwith appropriate vectors using recombinant DNA techniques. The vectorsand methods disclosed herein are suitable for use in host cells over awide range of eukaryotic organisms. This invention also encompasses thecells transformed with the novel replication and expression vectorsdescribed herein.

The Pnck gene made and isolated using the above methods can be directlyinserted into an expression vector, e.g., as in the Examples thatfollow, and inserted into a suitable animal or mammalian cell, such as amouse or mouse cell or that of a guinea pig, rabbit, simian cell, rat,or acceptable animal host cells, or into a human cell.

A variety of different gene transfer approaches are available to deliverthe Pnck gene into a target cell, cells or tissues. Among these areseveral non-viral vectors, including DNA/liposome complexes, andtargeted viral protein DNA complexes. In addition, the Pnck nucleic acidalso can be incorporated into a “heterologous DNA” or “expressionvector” for the practice of this invention. The term “heterologous DNA”is intended to encompass a DNA polymer, such as viral vector DNA,plasmid vector DNA, or cosmid vector DNA. Prior to insertion into thevector, it is in the form of a separate fragment, or as a component of alarger DNA construct, which has been derived from DNA isolated at leastonce in substantially pure form as described above, i.e., free ofcontaminating endogenous materials and in a quantity or concentrationenabling identification, manipulation, and recovery of the segment andits component nucleotide sequences by standard biochemical methods, forexample, using a cloning vector.

As used herein, “recombinant” is intended to mean that a particular DNAsequence is the product of various combination of cloning, restriction,and ligation steps resulting in a construct having a sequencedistinguishable from homologous sequences found in natural systems.Recombinant sequences can be assembled from cloned fragments and shortoligonucleotides linkers, or from a series of oligonucleotides.

As noted above, one means to introduce the nucleic acid into the cell ofinterest is by the use of a recombinant expression vector. “Recombinantexpression vector” includes vectors which are capable of expressing DNAsequences contained therein, where such sequences are operatively linkedto other sequences capable of effecting their expression. It is implied,although not always explicitly stated, that these expression vectorsmust be replicable in the host organisms either as episomes or as anintegral part of the chromosomal DNA. In sum, “expression vector” isgiven a functional definition, and any DNA sequence which is capable ofeffecting expression of a specified DNA sequence disposed therein isincluded in this term as it is applied to the specified sequence.

Suitable expression vectors include viral vectors, includingadenoviruses, adeno-associated viruses, retroviruses, cosmids andothers. Adenoviral vectors are a particularly effective means forintroducing genes into tissues in vivo because of their high level ofexpression and efficient transformation of cells both in vitro and invivo. Thus, in a preferred embodiment of the invention, a disease stateor cancer in a patient caused by, or related to, the expression of Pnck,is effectively treated by gene transfer by administering to that patientan effective amount of Pnck or an acceptable species-specific homologuethereof, wherein the gene is delivered to the patient by an adenovirusvector using recognized delivery methods.

The invention also relates to eukaryotic host cells comprising a vectorcomprising Pnck or a homologue thereof, particularly the humanhomologue, according to the invention. Such a cell is advantageously amammalian cell, and preferably a human cell, and can comprise saidvector in integrated form in the genome, or preferably in non-integrated(episome) form. The subject of the invention is also the therapeutic orprophylactic use of such vector comprising Pnck or a homologue thereof,particularly the human homologue, or eukaryotic host cell.

In addition, the present invention relates to a pharmaceuticalcomposition comprising as therapeutic or prophylactic agent a vectorcomprising Pnck or a homologue thereof, particularly the human homologueaccording to the invention, in combination with a vehicle, which isacceptable for pharmaceutical purposes. Alternately, it comprises anantisense Pnck molecule, or a Puck inhibitor molecule or suspected Puckinhibitor molecule.

The composition according to the invention is intended especially forthe preventive or curative treatment of disorders, such ashyperproliferative disorders and cancers, including those induced bycarcinogens, viruses and/or dysregulation of oncogene expression; or bythe activation of Pnck, or its homologue; or by expression oramplification of a presently unknown cell type, such as an epithelialcell, which is activated or transformed in the breast as a result of orrelated to Puck expression, or for which Pnck expression is anindicator. The treatment of cancer (before or after the appearance ofsignificant symptoms) is particularly preferred.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to reduce by at least about 15%, preferably by atleast 50%, more preferably by at least 90%, and most preferably completeremission of a hyperproliferative disease or cancer of the host.Alternatively, a “therapeutically effective amount” is sufficient tocause an improvement in a clinically significant condition in the host.In the context of the present invention, a therapeutically effectiveamount of the expression product of Pnck, or a homologue thereof,particularly the human homologue, is that amount which is effective totreat a proliferative disease or tumor or other cancerous condition, ina patient or host, thereby effecting a reduction in size or virulence orthe elimination of such disease or cancer. Preferably, administration orexpression of an “effective” amount of the expression product of Pnck ora homologue thereof, particularly the human homologue, resolves theunderlying infection or cancer.

A pharmaceutical composition according to the invention may bemanufactured in a conventional manner. In particular, a therapeuticallyeffective amount of a therapeutic or prophylactic agent is combined witha vehicle such as a diluent. A composition according to the inventionmay be administered to a patient (human or animal) by aerosol or via anyconventional route in use in the field of the art, especially via theoral, subcutaneous, intramuscular, intravenous, intraperitoneal,intrapulmonary, intratumoral, intratracheal route or a combination ofroutes. The administration may take place in a single dose or a doserepeated one or more times after a certain time interval.

The appropriate administration route and dosage vary in accordance withvarious parameters, for example with the individual being treated or thedisorder to be treated, or alternatively with the gene(s) of interest tobe transferred. The particular formulation employed will be selectedaccording to conventional knowledge depending on the properties of thetumor, or hyperproliferative target tissue and the desired site ofaction to ensure optimal activity of the active ingredients, i.e., theextent to which the protein kinase reaches its target tissue or abiological fluid from which the drug has access to its site of action.In addition, these viruses may be delivered using any vehicles usefulfor administration of the protein kinase, which would be known to thoseskilled in the art. It can be packaged into capsules, tablets, etc.using formulations known to those skilled in the art of pharmaceuticalformulation.

Dosages for a given host can be determined using conventionalconsiderations, e.g., by customary comparison of the differentialactivities of the subject preparations and a known appropriate,conventional pharmacological protocol. Generally, a pharmaceuticalcomposition according to the invention comprises a dose of the proteinkinase according to the invention of between 10⁴ and 10¹⁴,advantageously 10⁵ and 10¹³, and preferably 10⁶ and 10¹¹.

A pharmaceutical composition, especially one used for prophylacticpurposes, can comprise, in addition, a pharmaceutically acceptableadjuvant, carrier, fillers or the like. Suitable pharmaceuticallyacceptable carriers are well known in the art. Examples of typicalcarriers include saline, buffered saline and other salts, liposomes, andsurfactants. The adenovirus may also be lyophilized and administered inthe forms of a powder. Taking appropriate precautions not to denaturethe protein, the preparations can be sterilized and if desired mixedwith auxiliary agents, e.g., lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, and the like that do not deleteriously react with the activevirus. They also can be combined where desired with other biologicallyactive agents, e.g., antisense DNA or mRNA.

The compositions and methods described herein can be useful forpreventing or treating cancers of a number of types, including but notlimited to breast cancer, sarcomas and other neoplasms, bladder cancer,colon cancer, lung cancer, pancreatic cancer, gastric cancer, cervicalcancer, ovarian cancer, brain cancers, various leukemias and lymphomas.One would expect that any other human tumor cell, regardless ofexpression of functional p53, would be subject to treatment orprevention by the methods of the present invention, although theparticular emphasis is on mammary cells and mammary tumors. Theinvention also encompasses a method of treatment, according to which atherapeutically effective amount of the protein kinase, or a vectorcomprising same according to the invention is administered to a patientrequiring such treatment.

Also useful in conjunction with the methods provided in the presentinvention would be chemotherapy, phototherapy, anti-angiogenic orirradiation therapies, separately or combined, which maybe used beforeor during the enhanced treatments of the present invention, but will bemost effectively used after the cells have been sensitized by thepresent methods. As used herein, the phrase “chemotherapeutic agent”means any chemical agent or drug used in chemotherapy treatment, whichselectively affects tumor cells, including but not limited to, suchagents as adriamycin, actinomycin D, camptothecin, colchicine, taxol,cisplatinum, vincristine, vinblastine, and methotrexate. Other suchagents are well known in the art.

As described above, the agents encompassed by this invention are notlimited to working by any one mechanism, and may for example beeffective by direct poisoning, apoptosis or other mechanisms of celldeath or killing, tumor inactivation, or other mechanisms known orunknown. The means for contacting tumor cells with these agents and foradministering a chemotherapeutic agent to a subject are well known andreadily available to those of skill in the art.

As also used herein, the term “irradiation” or “irradiating” is intendedin its broadest sense to include any treatment of a tumor cell orsubject by photons, electrons, neutrons or other ionizing radiations.These radiations include, but are not limited to, X-rays,gamma-radiation, or heavy ion particles, such as alpha or betaparticles. Moreover, the irradiation may be radioactive, as is commonlyused in cancer treatment and can include interstitial irradiation. Themeans for irradiating tumor cells and a subject are well known andreadily available to those of skill in the art.

The protein kinase of the present invention can also be used to expressimmuno-stimulatory proteins that can increase the potential anti-tumorimmune response, suicide genes, anti-angiogenic proteins, and/or otherproteins that augment the efficacy of these treatments.

The present invention is further described in the following examples.These examples are provided for purposes of illustration only, and arenot intended to be limiting unless otherwise specified. The variousscenarios are relevant for many practical situations, and are intendedto be merely exemplary to those skilled in the art. These examples arenot to be construed as limiting the scope of the appended claims. Thus,the invention should in no way be construed as being limited to thefollowing example, but rather, should be construed to encompass any andall variations which become evident in light of the teaching providedherein.

EXAMPLES

The screening, RNA analyses, in situ hybridization and constructionsdescribed below are carried out according to the general techniques ofgenetic engineering and molecular cloning detailed in, e.g., Maniatis etal., (Laboratory Manual, Cold Spring Harbor, Laboratory Press, ColdSpring Harbor, N.Y. (1989)). The steps of PCR amplification follow knownprotocols, as described in, e.g., PCR Protocols-A Guide to Methods andApplications (ed., Innis, Gelfand, Sninsky and White, Academic PressInc. (1990)). Variations of such methods, so long as not substantial,are within the understanding of one of ordinary skill in the art.

Example 1 Protein Kinases Expressed During Mammary Development

To study the role of protein kinases in regulating mammary proliferationand differentiation, the following screen was designed to identifyprotein kinases expressed in the mammary gland and in breast cancer celllines. A reverse transcriptase (RT)-PCR cloning strategy was employedthat relied on the use of degenerate oligonucleotide primerscorresponding to conserved amino acid motifs present within thecatalytic domain of protein tyrosine kinases (Wilks et al., Gene85:67–74 (1989); Wilks et al., Proc. Natl. Acad. Sci. USA 86:1603–1607(1989)).

Cell Culture. Mammary epithelial cell lines were derived from mammarytumors or hyperplastic lesions that arose in mouse mammary tumor virus(MMTV)-c-myc, MMTV-int-2, MMTV-neu/NT, or MMTV-H-ras transgenic mice andincluded: the neu transgene-initiated mammary tumor-derived cell linesSMF, NAF, NF639, NF11005, and NK-2; the c-myc transgene-initiatedmammary tumor-derived cell lines 16MB9a, 8Ma1a, MBp6, M158, and M1011;the H-ras transgene-initiated mammary tumor-derived cell lines AC816,AC236, and AC711; the int-2 transgene-initiated hyperplastic cell lineHBI2; and the int-2 transgene-initiated mammary tumor-derived cell line1128 (Morrison et al., 1994). Additional cell lines were obtained fromATCC and included NIH3T3 cells and the nontransformed murine mammaryepithelial cell lines NMuMG and CL-S1. All cells were cultured underidentical conditions in DMEM medium supplemented with 10% bovine calfserum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 mg/mlstreptomycin.

Animals and Tissues. FVB mice were housed under barrier conditions witha 12-h light/dark cycle. The mammary glands from between 10 and 40age-matched mice were pooled for each developmental point. Mice forpregnancy points were mated at 4–5 weeks of age. Mammary gland harvestconsisted in all cases of the No. 3, 4, and 5 mammary glands. The lymphnode embedded in the No. 4 mammary gland was removed prior to harvest.Tissues used for RNA preparation were snap frozen on dry ice. Tissuesused for in situ hybridization analysis were embedded in O.C.T.embedding medium (10.24% polyvinyl alcohol; 4.26% polyethylene glycol),and frozen in a dry ice/isopentane bath. Developmental expressionpatterns for 13 kinases were confirmed using independent pools of RNA.Analysis of the developmental expression pattern for an additionalkinase using these independent pooled samples revealed a similarpregnancy-up-regulated expression pattern that differed with respect tothe day of pregnancy at which maximal up-regulation occurred.

Construction and Analysis of Kinase-Specific cDNA Libraries. RNAprepared from nine different sources was used as starting material forthe generation of kinase-specific cDNA libraries. Kinase-specific cDNAlibraries were constructed using mRNA prepared from the mammary glandsof mice at specified stages of development and from a panel of mammaryepithelial cell lines. Specifically, total RNA was prepared from themammary glands of either 5-week-old nulliparous female mice or parousmice that had undergone a single pregnancy followed by 21 days oflactation and 2 days of postlactational regression. Total RNA was alsoprepared from the seven mammary epithelial cell lines NMuMG, CL-S1,HBI2, SMF, 16MB9a, AC816, and 1128, described above (Leder et al., Cell45:485–495 (1986); Muller et al., 1988, 1990; Sinn et al., Cell49:465–475 (1987)).

Mammary tumors arising in each of these transgenic strains havepreviously been demonstrated to possess distinct and characteristichistopathologies that have been described as a large basophilic celladenocarcinoma associated with the myc transgene, a small eosinophiliccell papillary carcinoma associated with the H-ras transgene, a paleintermediate cell nodular carcinoma associated with the neu transgene,and a papillary adenocarcinoma associated with the int-2 transgene(Cardiff et al., 1993; Cardiff et al., Am. J. Pathol., 139:495–501(1991); Munn et al., Semin. Cancer Biol. 6:153–158 (1995)).

First-strand cDNA was generated from each of these nine sources of RNAusing the cDNA Cycle kit according to the manufacturer's directions(Invitrogen, San Diego, Calif.). These were amplified using degenerateoligonucleotide primers corresponding to conserved regions in kinasecatalytic subdomains VIb and IX. The degenerate primers, PTKIa(5′-GGGCCCGGATCCAC(A/C)G(A/G/C/T)GA(C/T)(C/T)-3′) SEQID NO:3, and PTKIIa(5′-CCCGGGGAATTCCA(A/T)AGGACCA(G/C)AC(G/A)TC-3′) SEQID NO:4, havepreviously been shown to amplify a conserved 200-bp portion of thecatalytic domain of a wide variety of tyrosine kinases (Hanks et al,1991; Wilks et al., 1989; Wilks, Methods Enzymol. 200:533–546 (1991)).In an effort to isolate a broad array of protein kinases, two additionaldegenerate oligonucleotide primers, BSTKIa(5′-GGGCCCGGATCC(G/A)T(A/G)CAC(A/C) G(A/G/C)GAC(C/T)T-3′) SEQID NO:5,and BSTKIIa (5′-CCCGGGGAATTCC(A/G)(A/T) A(A/G)CTCCA(G/C)ACATC-3′) SEQIDNO:6, were designed for use in this screen. These primers are alsodirected against subdomains VIb and IX, however, they differ innucleotide sequence. Restriction sites, underlined in the primersequences, were generated at the 5′ (ApaI and BamHI) and 3′ (XmaI andEcoRI) ends of the primer sequences.

Each cDNA source was amplified in three separate PCR reactions usingthree pairwise combinations of the PTKIa/PTKIIa, BSTKIa/BSTKIIa, andBSTKIa/PTKIIa degenerate primers to amplify first-strand cDNA from eachof the nine sources. Following 5-minutes denaturation at 95° C., sampleswere annealed at 37° C. for 1 min, polymerized at 63° C. for 2 min, anddenatured at 95° C. for 30 s for 40 cycles. The resulting ˜200-bp PCRproducts were purified from low-melting agarose (Boehringer MannheimBiochemicals), ligated into a T-vector (Invitrogen), and transformed inEscherichia coli. Following blue/white color selection, approximately 50transformants were picked from each of the 27 PCR reactions (3 reactionsfor each of nine cDNA sources) and were subsequently transferred togridded plates and replica plated. In total, 1450 transfornants wereanalyzed. Dideoxy sequencing of 100 independent transformants wasperformed, resulting in the identification of 14 previously describedtyrosine kinases.

In order to identify and eliminate additional isolates of these kinasesfrom further consideration, filter lifts representing the 1350 remainingtransfornants were hybridized individually to radiolabeled DNA probesprepared from each of the 14 initially isolated kinases. Hybridizationand washing were performed as described under final washing conditionsof 0.1×SSC/0.1% SDS at 70° C. that were demonstrated to preventcross-hybridization between kinase cDNA inserts (Marquis et al., Nat.Genet., 11:17–26 (1995). In this manner, 887 transformants (70% of thetransformants) were identified that contained cDNA inserts from the 14tyrosine kinases that had initially been isolated. Identifications madeby colony hybridization were consistent with those made directly by DNAsequencing.

The remaining 463 transformants were screened by PCR using T7 and SP6primers to identify those containing cDNA inserts of a length expectedfor protein kinases. One hundred seventy-two transformants were found tohave cDNA inserts between 150 and 300 bp in length. These were subclonedinto a plasmid vector and approximately 50 bacterial transformants fromeach of the 27 PCR reactions were replica plated and screened by acombination of DNA sequencing and colony lift hybridization in order toidentify the protein kinase from which each subcloned catalytic domainfragment was derived.

Individual clones were sequenced using the Sequenase® version 2 dideoxychain termination kit (U.S. Biochemical Corp., Cleveland, Ohio).Putative protein kinases were identified by the DFG(aspartate-phenylalanine-glycine) consensus located in catalyticsubdomain VI. DNA sequence analysis was performed using MacVector® 3.5(Oxford Molecular Group, Oxford, UK) and the NCBI BLAST server (Altshulet al. J. Mol. Biol., 215:403–410 (1990)).

RNA Preparation and Analysis. RNA was prepared by homogenization ofsnap-frozen tissue samples or tissue culture cells in guanidiniumisothiocyanate supplemented with 7 ml/ml 2-mercaptoethanol, followed byultra-centrifugation through cesium chloride as previously described(Marquis et al., 1995; Rajan et al., Dev. Biol., 184:385–401 (1997)).Poly(A)⁺ RNA was selected using oligo(dT) cellulose (Pharmacia,Piscataway, N.J.), separated on a 1.0% agarose gel (Seakem® LE,BioWhittaker Molecular Applications, Rockland, Me.), and passivelytransferred to a Gene Screen membrane (New England Nuclear, Boston,Mass.). Northern hybridization was performed as described using³²P-labeled cDNA probes corresponding to catalytic subdomains VI–IX ofeach protein kinase that were generated by PCR amplification of clonedcatalytic domain fragments (Marquis et al., 1995). In all casescalculated transcript sizes were consistent with values reported in theliterature.

In Situ Hybridization. In situ hybridization was performed as described(Marquis et al., 1995). Antisense and sense probes were synthesized withthe Promega (Madison, Wis.) in vitro transcription system using ³⁵S-UTPand ³⁵S-CTP from the T7 and SP6 RNA polymerase promoters of a PCRtemplate containing the sequences used for Northern hybridizationanalysis.

Discussion of Results. Analysis of the clones resulted in theidentification of 33 tyrosine kinases and 8 serine/threonine kinases(Table 1). The 19 receptor tyrosine kinases and 14 cytoplasmic tyrosinekinases that were isolated accounted for all but 33 of the 1056kinase-containing clones. The remaining clones were derived from 8serine/threonine kinases, 7 of which were represented by a single cloneeach, including each of the novel kinases isolated in this screen.Approximately half of the 41 kinases were isolated more than once, andmost of these were isolated from more than one tissue or cell line(Table 2, and data not shown). Eight (8) tyrosine kinases, includingJak2, Fgfr1, EphA2, Met, Igf1r, Hck, Jak1, and Neu, accounted for 830(79%) of all clones analyzed (Table 1). Conversely, 18 kinases (44%)were represented by a single clone each, suggesting that furtherscreening of cDNA libraries derived from these tissues and cell linesmay yield additional kinases.

TABLE 2 Protein Kinases Isolated from Mammary Glands and MammaryEpithelial Cell Lines. Receptor Nonreceptor tyrosine tyrosine kinaseskinases Serine/ threonine kinases Ax1/Ufo 6 c-Ab1 5 c-Akt1 1 EphA2 121Csk 46 Mlk1 1 1 EphA7 1 Ctk 1 Plk 26 EphB3 2 c-Fes 24 A-Raf 1 Egfr 1 Fyn7 SLK 1 Fgfr1 126 Hck 88 Flt3 1 Jak1 74 gflr 89 Jak2 150 InsR 1 Lyn 21c-Kit 2 Prkmk3 3 Novel Met 120 c-Src 23 kinases MuSK 1 Srm 1 Bstk1 1 Neu62 Tec 1 Bstk2 1 Ron 10 Tyk2 4 Bstk3 1 Ryk 1 Tie1 1 Tie2 27 Tyro10 2Tyro3 1 Note. Kinases are arranged by family and class. The number ofclones isolated for each kinase is shown on the right.

Three novel protein kinases were identified in this screen, designatedBstk1, 2, and 3. Each of these kinases contains the amino acid motifscharacteristic of serine/threonine kinases. Bstk2 and Bstk3 were eachisolated from the mammary glands of mice undergoing earlypostlactational regression. Bstk3 is most closely related tocalcium/calmodulin-dependent protein kinase I, and full-length isoformshave subsequently been identified in the mouse and rat (Yokokura et al.,Biochim. Biophys. Acta. 1338:8–12 (1997); Gardner et al., Genomics,63:279–288 (2000)). Characteristics and expression patterns for theremaining 43 protein kinases isolated by this screen are reported byChodosh et al., Develop. Biol. 219:259–276 (2000), which is hereinincorporated by reference in its entirety.

Example 2 Cloning, Characterization, and Chromosomal Location of Pnck

Recognizing the unique temporal and spatial expression pattern of Bstk3,it was renamed Pnck, for pregnancy-up-regulated, nonubiquitous CaMkinase. To isolate the full-length mRNA transcript from which Pnck(Bstk3) was derived, the initial 204-bp RT-PCR product was used toscreen a murine brain cDNA library.

Cloning of a Full-Length Pnck cDNA. The original catalytic domainfragment, Bstk3, from Example 1, corresponding to nucleotides 501 to 704of full-length Pnck, was used to screen 5×10⁵ lambda phage plaques froman oligo(dT)-primed murine brain cDNA library according to standardprotocols (CPMB). Primary screening yielded a total of 73 clones ofvarying hybridization intensity that were positive on duplicate filters.Ten (10) clones with medium to high hybridization intensity were plaquepurified, and plasmids were liberated by in vivo excision according tothe manufacturer's instructions (Stratagene, La Jolla, Calif.). Sequenceanalysis of 5 of these clones revealed a high level of homology toCaMKI. The remaining 5 clones were found to encode portions of Pnck asdetermined by overlapping sequence identity to one another and to Bstk3.

Two clones were not studied further since one clone was chimeric and asecond clone contained only partial Pnck sequence.

Three nonchimeric cDNA clones, U7, V1, and Q3, ranging from 1455 to 1554nucleotides in length were isolated and completely sequenced byautomated sequencing using an ABI Prism 377 DNA sequencer. Nucleotidesequence alignment revealed that the three clones differed only in theirrespective 5′-UTR sequences. The sequence of each cDNA clone containedthe entire 204-bp RT-PCR fragment, Bstk3, as well as a 1029-nucleotideopen reading frame (ORF) and a 420-bp 3′-UTR possessing apolyadenylation signal and poly(A) tract (FIG. 1).

Inspection of the nucleotide sequence surrounding the putativeinitiation codon at nucleotide 105 of the longest clone, U7, revealsmatches with the Kozak translational initiation consensus sequence atpositions −1, −3, −5, and −6 (Kozak, Nucleic Acids Res., 15:8125–8132(1987); Kozak, Cell Biol., 115:887–903 (1991), demonstrating that thepredicted initiation codon is capable of functioning as a translationinitiation site. Since clone U7 contains multiple in-frame terminationcodons upstream of this putative initiation codon, these findingsconfirmed that the entire Pnck coding sequence had been isolated.

Conceptual translation of the Pnck ORF yielded a 343-amino-acidpolypeptide of predicted molecular mass 38.6 kDa.

The full-length Pnck cDNA sequence corresponding to the clone with thelongest 5′-UTR, U7, was deposited with the GenBank® database (AccessionNo. AF181984).

Sequence Analysis. Sequence analysis, including prediction of openreading frames, calculation of predicted molecular weights, multiplesequence alignment, and phylogenetic analysis, was performed usingMacVector® (Oxford Molecular Group, Oxford, UK), ClustalW (Thompson etal., Nucleic Acids Research, 22:4673–4680, 1994), ClustalX (Thompson etal., Nucleic Acids Res., 24:4876–4882 (1997)), and DendroMaker 4.0(Tadashi Imanishi, Center for Information Biology, National Institute ofGenetics). Pairwise and multiple sequence alignments of kinase catalyticdomains I-XI were performed using the ClustalW alignment program.Calculations were made using the BLOSUM series (Henikoff et al., ProcNatl Acad Sci USA 89:10915–10919 (1992)) with an open gap penalty of 10,an extended gap penalty of 0.05, and a delay divergent of 40%.Phylogenetic calculations with the same parameters were performed usingthe ClustalX multisequence alignment program.

Tissue Preparation. FVB mouse embryos were harvested at specified timepoints following timed matings. Day 0.5 postcoital was defined as noonof the day on which a vaginal plug was observed. Tissues used for RNApreparation were harvested from 15- to 16-week-old virgin mice andsnap-frozen on dry ice. Tissues used for in situ hybridization analysiswere embedded in O.C.T. compound.

RNA Analysis. To determine whether the lengths of the cDNA clonesencoding Pnck were consistent with the size of the Pnck mRNA transcript,Northern hybridization was performed. RNA was prepared by homogenizationof snap-frozen tissue samples in guanidinium isothiocyanate supplementedwith 7 ml/ml 2-mercaptoethanol followed by ultracentrifugation throughcesium chloride as in Example 1. Poly(A)⁺ RNA from adult murine brainwas selected using oligo(dT) cellulose (Pharmacia), separated on a 1% LEagarose gel, and passively transferred to a Gene Screen membrane (NEN),again as in Example 1. Northern hybridization was performed using 4 μgsamples of the adult murine brain poly(A)⁺ RNA hybridized with a 3′-UTR³²P-labeled cDNA probe encompassing nucleotides 1135 to 1509 of Pnck,generated by random-primed labeling (Boehringer Mannheim Biochemicals,Indianapolis, Ind.).

In vitro transcription/translation reactions were performed using³⁵S-labeled methionine-labeled reticulocyte lysates with a full-lengthPnck cDNA clone (V1, U7, or Q3) or a cDNA plasmid encoding an unrelatedkinase (−) as a negative control (FIG. 2B). IVT reactions were resolvedon a 10% SDS-PAGE gel.

Due to potential cross-hybridization between Pnck and homologous CaMkinase family members, Southern hybridization was used to confirm thespecificity of a probe generated from the 3′-UTR of Pnck (data notshown). Southern hybridization analysis was performed on a zoo-blot(Clontech, Palo Alto, Calif.) hybridized with a ³²P-labeled cDNA probecorresponding to nucleotides 1321 to 1509 from the 3′-UTR of Pnck.Hybridization and washes were performed according to the manufacturer'sdirections (Clontech).

Consistent with the lengths of the isolated Pnck cDNA clones, thisanalysis revealed an mRNA transcript approximately 1.6 kb in length(FIG. 2A), set forth as SEQID NO:1 (nucleic acid) and SEQID NO:2 (aminoacid), respectively. A single band was detected in genomic DNA from bothmouse and rat, confirming that, under these conditions, thisPnck-specific 3′-UTR probe recognizes a single locus. Ribonucleaseprotection analysis was performed as described (Marquis et al., 1995).Body-labeled antisense riboprobes were generated using linearizedplasmids containing nucleotides 1321 to 1509 of Pnck and 1142 to 1241 ofβ-actin (GenBank® Accession No. X03672) using [α-³²P]UTP and the Promegain vitro transcription system with T7 polymerase. A β-actin antisenseriboprobe was added to each reaction as an internal control. Probes werehybridized with RNA samples at 58° C. overnight in 50% formamide/100 mMPIPES (pH 6.7) (Piperazine-N,N′-bis[2-ethanesulfonicacid];1,4-Piperazinediethane sulfonic acid). Hybridized samples weredigested with RNase A and T1, purified, electrophoresed on a 6%denaturing polyacrylamide gel, and subjected to autoradiography.

In Vitro Transcription and Translation. To confirm the coding potentialof the Pnck ORF, in vitro transcriptions and translations were performedin the presence of ³⁵S-methionine using each of the three Pnck cDNAclones as template. In vitro transcription and translation wereperformed on 1 μg of plasmid DNA using rabbit reticulocyte lysates inthe presence of ³⁵S-labeled methionine according to the manufacturer'sinstructions (TNT kit, Promega) and 1 mg of template consisting of afull-length Pnck cDNA clone (V1, U7, or Q3) or a cDNA plasmid encodingan unrelated kinase as a negative control. Completed reactions wereelectrophoresed on a 10% SDS-PAGE gel, then subjected toautoradiography. In each case, incubation of plasmid DNA withreticulocyte lysate yielded a single labeled polypeptide species ofapproximately 38 kDa, consistent with the predicted Pnck ORF (FIG. 2B).

Interspecific Mouse Backcross Mapping to Determine ChromosomalLocalization. The chromosomal location of murine Pnck was determined byinterspecific backcross analysis using progeny derived from matings of(C57BL/6JX M. spretus) F₁ females and C57BL/6J males as described byCopeland et al., Trends Genet., 7:113–118 (1991)). A total of 205 N₂mice were used to map the Pnck locus. DNA isolation, restriction enzymedigestion, agarose gel electrophoresis, Southern blot transfer, andhybridization were performed essentially as described by Jenkins et al.,J. Virol., 43:26 (1982). All blots were prepared with Hybond-N⁺ nylonmembrane (Amersham, Arlington Heights, Ill.).

The probe, a 375-bp fragment corresponding to nucleotides 1135 to 1509of mouse Pnck cDNA, was labeled with [α-³²P]dCTP using anick-translation labeling kit (Boehringer Mannheim Biochemicals);washing was performed at a final stringency of 1.0×SSCP, 0.1% SDS at 65°C. A fragment of 13.0 kb was detected in PstI-digested C57BL/6J DNA, anda fragment of 5.1 kb was detected in PstI-digested M. spretus DNA. Thepresence or absence of the 5.1-kb PstI M. spretus-specific fragment wasfollowed in backcross mice. (A description of the probes and RFLPs for aloci linked to Pnck including Tnfsf5, Il1rak, and Ar has been reportedpreviously (Centanni et al., Mamm. Genome., 9:340–341 (1998)).

Recombination distances were calculated using Map Manager, version 2.6.5(Manly et al., Mammalian Genome, 4:303–313 (1993)). Gene order wasdetermined by minimizing the number of recombination events required toaccount for the allele distribution patterns.

This interspecific backcross mapping panel has been typed for over 2800loci that are well distributed among all the autosomes as well as the Xchromosome. C57BL/6J and M. spretus DNAs were digested with severalenzymes and analyzed by Southern blot hybridization for informativerestriction fragment length polymorphisms (RFLPs) using a cDNA probespecific for the 3′-UTR of Pnck. The 5.1-kb PstI M. spretus RFLP (above)was used to follow the segregation of the Pnck locus in backcross mice.The mapping results indicated that Pnck is located in the central regionof the mouse X chromosome linked to Tnfsf5, Il1rak, and Ar.

Although 106 mice were analyzed for every marker and evaluated in asegregation analysis (not shown), up to 142 mice were typed for somepairs of markers. Each locus was analyzed in pairwise combinations forrecombination frequencies using the additional data. The ratios of thetotal number of mice exhibiting recombinant chromosomes to the totalnumber of mice analyzed for each pair of loci and the most likely geneorder are centromere−Tnfsf5−15/137−Pnck−0/134−Il1rak−9/142−Ar.

The recombination frequencies expressed as genetic distances incentimorgans ± the standard error are −Tnfsf5−11.0±2.7−(Pnck,Il1rak)−6.3±2.0−Ar. No recombinants were detected between Pnck andIl1rak in 134 animals typed in common, suggesting that the two loci arewithin 2.2 cM of each other (upper 95% confidence limit). In addition,the interspecific map of the X chromosome was compared with a compositemouse linkage map that reports the map location of many uncloned mousemutations (provided by Mouse Genome Database,.

Pnck maps to a region of the composite map that lacks uncloned mousemutations (data not shown). The central region of the mouse X chromosomeshares a region of conserved homology with the long arm of the human Xchromosome (summarized in FIG. 3). In particular, Il1rak has been mappedto Xq28. Therefore, in light of the close linkage between Il1rak andPnck in mouse, it is determined that the human homologue of Pnck willmap to Xq28, as well.

Analysis of Pnck mRNA Expression In Situ Hybridization. As part ofdetermining the biological role of Pnck, the developmental expressionpattern of Pnck mRNA was analyzed during murine embryogenesis. Northernhybridization analysis was performed on poly(A)⁺ RNA isolated fromembryos during early, mid-, and late gestation using a Pnck-specificprobe (FIG. 4A). Compared to mRNA expression levels in earlyembryogenesis, steady-state Pnck mRNA levels are markedly up-regulatedin the embryo during midgestation and remain elevated through embryonicday 18.5.

To investigate the spatial pattern of Pnck expression during fetaldevelopment, in situ hybridization analysis was performed (by themethods described in Example 1) on embryonic sections at day 14.5 ofgestation. ³⁵S-labeled Pnck antisense and sense probes were synthesizedwith the Promega in vitro transcription system using ³⁵S-UTP and ³⁵S-CTPfrom the T7 and SP6 RNA polymerase promoters of a PCR templatecontaining sequences corresponding to nucleotides 1135 to 1509 of Pnck(FIG. 4B). No signal-over-background was detected in serial sections ofbone, basal telen-cephalon, fourth ventricle, liver, lung, lateralventricle, stomach, trigeminal ganglion or whisker hair folliclehybridized with a sense Pnck probe.

This analysis revealed tissue-specific expression of Pnck in the embryoat midgestation with highest levels of expression detected in developingbone, the outer lining of the stomach, and the developing centralnervous system, including periventricular regions and the trigeminalganglion.

The expression profile of Pnck in tissues of the adult mouse wasdetermined by RNase protection analysis (FIG. 5A), using 30 mg RNAisolated from the indicated murine tissues using antisense RNA probesspecific for Pnck. β-actin was used as an internal control, and tRNA wasused as a negative control for nonspecific hybridization. Although Pnckexpression in the embryonic and adult mouse is highest in brain,moderate to low levels of Pnck expression are detected in hormonallyresponsive tissues such as uterus, ovary, testis, and mammary gland, aswell as in other tissues such as stomach, heart, and skeletal muscle.Lower, but detectable, levels of Pnck expression were observed inthymus, spleen, duodenum, and lung.

Finally, the spatial expression pattern of Pnck in adult murine tissueswas determined by in situ hybridization analysis in brain (FIGS. 5B, 5C,5F, 5G, 5J, 5K), testis (FIGS. 5D, 5E), ovary (FIGS. 5H, 5I), andprostate (FIGS. 5L, 5M), hybridized with a ³⁵S-labeled Pnck antisenseprobe. Interestingly, within expressing tissues Pnck mRNA was detectedin only a subset of cells. In the brain, Pnck expression is highest inthe dentate gyrus and CA1–3 regions of the hippocampus (FIGS. 5B, 5C,5F, and 5G). Pnck is also expressed at relatively high levels in thecortex and is markedly heterogeneous with highly expressing cells foundadjacent to nonexpressing cells (FIGS. 5J and 5K). Pnck is expressedthroughout the ovary, but is preferentially localized in the thecal celllayers immediately surrounding the corpora lutea (FIGS. 5H and 5I). Inthe testis, Pnck is expressed at high levels in mature spermatidsresiding at the center of seminiferous tubules and, to a lesser extent,in cells located adjacent to the basement membrane (FIGS. 5D and 5E).Finally, in the dorsolateral prostate, Pnck mRNA is detected in astromal layer of cells immediately surrounding the prostatic epithelialducts. As in other tissues, Pnck expression in this compartment isspatially heterogeneous (FIGS. 5L and 5M).

No signal-over-background was detected in serial sections hybridizedwith a sense Pnck probe.

Example 3 Analysis of Spatial and Temporal Profile of Pnck Expression

To examine the potential role of Pnck in mammary development, thetemporal profile of Pnck expression was analyzed during the postnataldevelopment of the murine mammary gland.

Animal and Tissue Preparation. FVB mice were housed under barrierconditions with a 12-hour light/dark cycle. After sacrifice at theindicated developmental time points, the #3, 4, and 5 mammary glandswere harvested. For RNA analysis, the lymph node embedded in mammarygland #4 was removed prior to harvest. Timed matings were set up, suchthat all mice were sacrificed at ˜16 weeks of age for comparison toadult nulliparous females. Day 0.5 postcoitus was, as in Example 2,defined as noon of the day on which a vaginal plug was observed. Timepoints at day 2 and day 7 of regression were obtained after removingpups at day 9 of lactation. Time points at day 28 of regression wereobtained after 21 days of lactation. Tissues from 10 to 20 mice werepooled for each developmental time point.

Tissues used for RNA analysis were snap frozen on dry ice. Tissues usedfor in situ hybridization analysis were embedded in O.C.T. compound.

Tissue Culture. Murine cells were cultured in DMEM medium supplementedwith 10% bovine calf serum, 2 mM L-glutamine, 100 units/ml penicillin,and 100 μg/ml streptomycin. Human cell line lines were cultured in thesame medium with the addition of 5 μg/ml insulin.

Transformed murine mammary epithelial cell lines were derived fromtumors or hyperplastic lesions that arose in transgenic mice engineeredto express different oncogenes under the control of the MMTV longterminal repeat (MMTV-LTR). Cell lines from MMTV-c-myc, MMTV-int-2/Fgf3,MMTV-neu/NT, or MMTV-H-ras transgenic mice have been describedpreviously (Morrison et al., 1994). NIH 3T3, NMuMG, and CL-S1 murinecells, as well as human breast tumor cell lines, were obtained fromAmerican Type Culture Cells. HC11 cells were from J. Rosen (BaylorCollege of Medicine, Houston, Tex.).

Actively growing cells were harvested at ˜70% confluence. Confluentcells were re-fed daily and harvested 3 days after confluence. For serumstarvation experiments, subconfluent cells were maintained in 0.1% serumfor 2 days prior to re-feeding in 10% bovine calf serum and harvested atthe indicated time points.

RNA Analysis. RNA was prepared by homogenization of snap-frozen tissuesamples or tissue culture cells in guanidinium isothiocyanatesupplemented with 7 μl/ml 2-mercaptoethanol, followed byultracentrifugation through cesium chloride as described previously(Rajan et al., 1997; Marquis et al., 1995). Samples of 40 μg of totalRNA isolated from mammary glands at selected developmental time points(see FIG. 7) were hybridized to ³²P-labeled antisense riboprobesspecific for the 3′ untranslated region of Pnck, or for β-actin (FIG.6A). Poly(A)⁺ RNA was selected using oligo(dT) cellulose (Pharmacia).Pnck expression was quantified and normalized to β-actin expression tocorrect for dilutional effects of large scale increases in milk proteingene expression during late pregnancy and lactation. Expression levelswere compared with matched 16-week old adult virgin animals (FIG. 6B).

For Northern hybridization analysis, RNA was separated on a 1% LEagarose gel and passively transferred to a Gene Screen membrane (DuPontNEN). Hybridization was performed as described using a random primed,³²P-labeled cDNA probe encompassing nucleotides 1355–1529 of c-myc(GenBank® accession no. X01023), nucleotides 589–1287 of cytokeratin 18(GenBank® accession No. M11686), or a 1.2-kb fragment containing theentire open reading frame of cyclin D3 (Marquis et al., 1995).

RNase protection analysis was performed as described (Marquis et al.,1995). Body-labeled antisense riboprobes were generated using [α-³²P]UTPand the Promega in vitro transcription system with T7 polymerase incombination with linearized plasmids containing nucleotides 1142–1241 ofβ-actin, nucleotides 911–1056 of Gapdh (glycelaldehyde-3-phosphatedehydrogenase) locusi (GenBank® accession No. M32599), nucleotides1321–1509 of murine Pnck (GenBank® accession No. AF181984), or a regionof human PNCK corresponding to nucleotides 538–842 of murine Pnck.Riboprobes were hybridized with RNA samples overnight at 58° C. in 50%formamide/100 mM PIPES (pH 6.7).

Pnck expression was normalized to β-actin expression to control fordilutional effects resulting from the massive increases in milk proteingene expression that occur during late pregnancy and lactation (FIG.6B). Hybridized samples were digested with RNase A and T1, purified,electrophoresed on a 6% denaturing polyacrylamide gel, and subjected toautoradiography (XAR-5). β-actin or Gapdh antisense riboprobes wereadded to each reaction as an internal control. As a negative control,riboprobes were hybridized with tRNA and processed in parallel.

Heterogeneous Expression Shown by In Situ Hybridization. To determinewhether pregnancy-induced changes in Pnck mRNA expression levelsrepresent global changes in expression throughout the mammary gland orchanges within a subpopulation of cells, quantitative in situhybridization analysis was performed as described (Marquis et al.,1995). Antisense and sense riboprobes were synthesized with the Promegain vitro transcription system using ³⁵S-UTP and ³⁵S-CTP from the T7 andSP6 RNA polymerase promoters of a PCR template containing sequencescorresponding to nucleotides 1135–1509 of Pnck. Pnck mRNA expressionlevels were found to be low and relatively constant in nulliparousanimals between 2 and 16 weeks of age, a period that encompasses ductalmorphogenesis (FIGS. 7A–7F). In contrast, a 2-fold up-regulation of Pnckexpression was observed early in pregnancy as compared with age-matchednulliparous animals. Consistent with the RNase protection results, insitu hybridization confirmed that Pnck expression remained elevatedduring mid-pregnancy and attained maximal levels of expression (5-fold)late in pregnancy (FIGS. 7G–7H), concomitant with the cessation ofproliferation and terminal differentiation of the alveolar epithelium.Pnck expression levels returned to baseline during lactation and earlypostlactational regression (FIGS. 7I–J).

Although throughout postnatal development Pnck expression was detectedonly in the mammary epithelium, it was strikingly heterogeneous duringpregnancy, with highly expressing cells located adjacent to cells inwhich Pnck expression was low or undetectable. The spatial heterogeneityof Pnck expression was most marked during late pregnancy, at which timeonly a small fraction of epithelial cells was observed to express Pnckat high levels. The heterogeneous spatial pattern of Pnck expressiondiffers from that observed for other protein kinases, as well as forgenes such as cytokeratin 18, Gapdh, and β-actin (Chodosh, et al.,2000).

Notably, steady-state levels of Pnck mRNA were higher in the mammaryglands of parous animals after 4 weeks of postlactational involution ascompared with age-matched nulliparous animals. Moreover, as verified byquantitative in situ hybridization analysis, normalization of geneexpression to b-actin expression provides a more accurate assessment ofchanges in gene expression on a per cell basis than normalization solelyto the amount of RNA assayed (FIG. 7).

Pnck Expression in Vitro. The observation that Pnck expression peakslate in pregnancy as alveolar epithelial cells exit the cell cycle andundergo terminal differentiation suggested that Pnck mRNA expression maybe inversely related to mammary epithelial proliferation. To investigatethis possibility, Pnck mRNA levels were analyzed in activelyproliferating or confluent mammary epithelial cell lines (FIG. 8A).³²P-labeled antisense riboprobes specific for Pnck or Gapdh werehybridized with 30 μg of total RNA isolated from the following celllines: 16 MB9a, M158 and HB12, while either actively growing (Act) or 3days after confluence (Con).

This analysis revealed that steady-state levels of Pnck mRNA were anaverage of 3.7-fold higher in confluent cells compared with activelyproliferating cells (Student's t test, P, 0.01).

To distinguish whether this increase in Pnck expression was attributableto decreased proliferation or to the establishment of cell-cell contactsin confluent cells, Pnck expression levels were analyzed in subconfluentserum-starved 16MB9a mammary epithelial cells as they reentered the cellcycle after re-feeding (FIG. 8B). 30 μg of total RNA isolated from cellsat each time point were hybridized with ³²P-labeled antisense riboprobesspecific for Pnck, or for β-actin at 0, 0.5, 1, 2, 4, 6 and 9 hours,respectively, after re-feeding. Consistent with the up-regulation ofPnck expression observed in confluent cells, re-feeding of serum-starvedcells resulted in a rapid decrease in Pnck expression that began within1 hour, and reached a nadir at 4 hours after re-feeding. Identicalresults were observed in a second mammary epithelial cell line (data notshown).

Pnck Expression in Transgenic Mammary Tumor Cell Lines. To examine thepotential role of Pnck in mammary tumorigenesis and to investigate thehypothesis that Pnck is expressed in an epithelial cell subtype in themammary gland, Pnck mRNA expression was examined in a panel of mammaryepithelial cell lines derived from independent adenocarcinomas arisingin MMTV transgenic mice expressing the neu/NT, c-myc, H-ras, orint-2/Fgf3 oncogenes in the mammary gland epithelium (Morrison et al.,1994; FIG. 9. The cell lines used were: NIH-3T3 fibroblast,nontransformed (Non-Tx): Lane 1, NMuMG, Lane 2, HC11, and Lane 3, CL-S1.MMTV-int-2/Fgf3: Lane 4, HBI2; and Lane 5, 1128. MMTV-c-myc: Lane 6,8MA1a; Lane 7, MBp6; Lane 8, M1011; Lane 9, M158; and Lane 10, 16MB9a.MMTV-neu: Lane 11, SMF; Lane 12, NaF; Lane 13, NF639; Lane 14, NF11005;and Lane 15, NK-2. MMTV-H-ras: Lane 16, AC816; Lane 17, AC711; and Lane18, AC236. The poly(A)⁺ RNA beneath the 28S rRNA band was used as aloading control.

RNase protection analysis was performed on 6 μg of poly(A)⁺ RNA isolatedfrom he actively growing murine cell lines hybridized with a ³²P-labeledantisense riboprobe specific for the 3′ untranslated region of Pnck(FIG. 9, top panel). Northern analysis was performed on 6 μg of poly(A)⁺RNA using ³²P-labeled cDNA probes specific for c-myc (FIG. 9, middlepanel) or cyclin D3 (FIG. 9, bottom panel). All cell lines wereproliferating at similar rates when harvested as evidenced by theirsimilar levels of cyclin D3 mRNA expression.

Pnck expression was not detected in NIH 3T3 fibroblasts, consistent withits epithelial-specific pattern of expression in the mammary gland invivo. Interestingly, however, Pnck was expressed in all seven cell linesderived from mammary tumors or hyperplasias arising in MMTV-c-myc and inMMTV-int-2/Fgf3 transgenic mice.

In contrast, Pnck expression was undetectable in the eight cell linesderived from mammary tumors arising in MMTV-neu and MMTV-H-rastransgenic mice, despite the fact that RNase protection analysis wasperformed using poly(A)⁺ RNA. Similarly, Pnck expression was notdetected in any of the three nontransformed mammary epithelial celllines examined, including confluent or differentiating HC11 cells (FIG.9; HC11 data not shown).

Analysis of the expression of 40 other protein kinases identified in thescreen indicated that this particular oncogene-associated pattern ofexpression is unique to Pnck (Chodosh et al., 2000, Gardner et al.,2000). Of note, the upper band observed in MMTV-c-myc-derived cell lineswas found to correspond to c-myc transgene expression. However, Pnckexpression did not appear to correlate with absolute levels of eitherendogenous c-myc or c-myc transgene expression (FIG. 9). Consequently,although c-myc or int-2/Fgf3 could directly up-regulate Pnck expression,the lack of correlation between Pnck expression and c-myc expression inmammary tumor cell lines, along with the punctate expression of Pnck invivo, raises the possibility that the oncogene-associated expression ofPnck more likely results from the preferential transformation of aPnck-expressing cell type by c-myc, and that the oncogene-restrictedpattern of Pnck expression may not be the result of c-myc-inducedactivation of Pnck transcription. Thus, these morphological differencesappear to either result from the activation of unique downstreampathways or from the preferential transformation of different epithelialcell types by these oncogenes.

Example 4 PNCK Expression in Human Breast Tumor Cell Lines and PrimaryBreast Tumors

To investigate the potential involvement of Pnck, or a cell type inwhich Pnck is expressed, in human mammary carcinogenesis, PNCKexpression levels were determined in a panel of human breast cancertumor cell lines (FIG. 10). An RNase protection analysis was performedusing 30 μg of total RNA isolated from actively growing human breasttumor cell lines hybridized with a ³²P-labeled antisense riboprobespecific for PNCK, or for β-actin. As a negative control, tRNA was usedfor comparison. The cell lines used were: Lane 1, 184B5; Lane 2, 2 MT-2;Lane 3, BT-20; Lane 4, BT-474; Lane 5, BT-549; Lane 6, HBL-100; Lane 7,MDA-MB-157; Lane 8, MDA-MB-231; Lane 9, MDA-MB-361; Lane 10, MDA-MB-435;Lane 11, MDA-MB-436; Lane 12 MDA-MB-453; Lane 13, MDA-MB-468; Lane 14,SK-BR-3; Lane 15, ZR-75-1; Lane 16, MCF-10; Lane 17, MCF-10A; and Lane18, Hs 578T.

Similar to the wide range of Pnck expression observed in the murinemammary epithelium and in murine mammary tumor cell lines, PNCKexpression was detected in only a subset of human breast tumor celllines (See FIG. 10). High levels of PNCK expression were observed in 3of 18 breast tumor cell lines. Eight cell lines expressed low, butdetectable levels of PNCK, whereas no PNCK expression was detected inthe remaining seven cell lines. As seen in the murine mammary tumor celllines, PNCK expression levels did not correlate with c-MYC expression(data not shown).

The heterogeneous pattern of Pnck expression observed in vitro in bothmurine and human breast tumor cell lines suggested the possibility thatPNCK-expressing and PNCK-nonexpressing breast tumor types might exist.To test this hypothesis directly, an RNase protection analysis was usedto quantify PNCK mRNA expression levels in a panel of human breasttissue samples.

RNA was isolated from 12 benign breast tissue samples and from 23primary breast tumors obtained after surgery. An RNase protectionanalysis was performed using 10 μg of total RNA hybridized with a³²P-labeled antisense riboprobe specific for PNCK, or for β-actin asindicated in FIG. 11A. Northern hybridization analysis was performed onthe same RNA samples using 3 μg of total RNA hybridized with a³²P-labeled cDNA probe specific for cytokeratitn 18 (CK18) (FIG. 11A).PNCK, β-actin, and CK18 expression levels were quantified byphosphorimager analysis, and PNCK expression levels were normalized toCK18 for each sample.

PNCK expression levels in breast tumors were then compared with benigntissue as shown in FIG. 11B. PNCK expression levels for the samplesshown in FIG. 11A were normalized either to β-actin or to CK18.Normalized PNCK expression levels in the benign tissues was set equal to1.0. The means of each distribution are shown in FIG. 11B. PNCK/β-actinand PNCK/CK18 expression in tumors was compared with benign tissue.

FIG. 11C presents a histogram of individual PNCK expression levelsnormalized to CK18 for the primary breast tumors and benign breasttissue samples shown in FIG. 11A. PNCK and cytokeratin 18 expressionlevels were quantified by phosphorimager analysis. PNCK expression foreach sample was normalized to CK18 expression, and the averageexpression in benign samples was set equal to 1.0. The values of thefold changes relative to the mean PNCK/CK18 expression level observedfor benign breast tissue are shown in FIG. 11C.

This analysis revealed two interesting aspects of the pattern of PNCKexpression in breast tumors compared with benign tissue: (a) PNCK isexpressed at significantly higher levels in breast tumors compared withbenign tissue; and (b) PNCK expression in human tumors is markedlyheterogeneous. Statistical analysis of the examined PNCK expressionlevels indicated that when normalized to β-actin expression, PNCKexpression in human primary breast cancers is ˜5-fold higher than inbenign breast tissue (Student's t test, P=0.01; FIG. 11B). However,because PNCK expression in the mammary gland is epithelial specific, andtumors typically have a higher epithelial content than benign breasttissue, PNCK expression was also normalized to expression of theepithelial-specific marker, cytokeratin 18, (CK18), to control for theincreased epithelial cell content in the tumors (FIG. 11B).Nevertheless, even after normalization to CK18 expression, PNCKexpression levels were found to be three times (3×) higher in humanprimary breast tumors than in benign tissue (t test, P 5 0.039).

Formally, the increase in PNCK expression levels in breast tumorscompared with benign tissue could have resulted either from increasedexpression among all tumors or from increased expression in a subset oftumors. However, analysis of the distribution of PNCK expression amongthe 23 ductal carcinomas studied revealed a wide range of PNCKexpression levels, in contrast to the relatively similar levels of PNCKexpression observed among benign breast tissue samples. Notably, themode for the benign and tumor distributions was the same (FIGS. 11A and11C). Indeed, examination of the histogram representing CK18-normalizedPNCK expression levels revealed that 8 of the 23 primary breast tumorsanalyzed expressed PNCK at levels greater than 3 standard deviationsabove the mean observed for benign samples (FIG. 11C). This differenceis highly significant because no tumors would have been predicted toexpress PNCK at these levels if the distribution of PNCK expression intumors was similar to that observed in benign tissues. Even morestrikingly, four (4) breast tumors were found to express PNCK atlevels >10 standard deviations above the mean observed for benigntissues. Together, these data demonstrate that PNCK is overexpressed inhuman primary breast cancers, when compared with benign tissue, and thatthis observed increase is attributable to high levels of PNCK expressionin a subset of breast tumors.

Example 5 PNCK Expression in Human Primary Ovarian and Colon Tumors

To investigate the potential involvement of Pnck, or a cell-type inwhich Pnck is expressed, in human ovarian and colon carcinogenesis, PNCKexpression levels were determined in a panel of human primary ovarianand colon cancers along with benign tissue samples from each of theseorgans. An RNase protection analysis was performed, as above, using 30μg of total RNA isolated from tumors hybridized with a ³²P-labeledantisense riboprobe specific for PNCK or for β-actin. As a negativecontrol, tRNA was used for comparison.

RNA was isolated from 16 benign ovarian tissue samples and from 22primary ovarian tumors obtained after surgery. An RNase protectionanalysis was performed using 10 μg of total RNA hybridized with a³²P-labeled antisense riboprobe specific for PNCK or for β-actin. PNCKand β-actin expression levels were quantified by phosphorimageranalysis, as above, and PNCK expression levels were normalized toβ-actin for each sample. PNCK expression levels in ovarian tumors werecompared with benign tissue. Normalized PNCK expression levels in thebenign tissues was set equal to 1.0. This analysis demonstrated thatPNCK is expressed in ovarian tumors at a level that is 4.1-fold higherthan in benign ovarian tissue (p=0.011). Further analysis of PNCKexpression as a function of ovarian tumor grade revealed that PNCKexpression correlates positively with tumor grade, withpoorly-differentiated tumors exhibiting higher levels of PNCK expressionthan moderately differentiated tumors, and moderately-differentiatedtumors exhibiting higher levels of PNCK expression thanwell-differentiated tumors.

In a similar manner, RNA was isolated from 17 benign colon tissuesamples and from 24 paired primary colon tumors obtained after surgery(e.g., benign samples were taken from the same patient as the tumorsamples). An RNase protection analysis was performed using 10 μg oftotal RNA hybridized with a ³²P-labeled antisense riboprobe specific forPNCK or for β-actin. PNCK and β-actin expression levels were quantifiedby phosphorimager analysis, and PNCK expression levels were normalizedto β-actin for each sample. PNCK expression levels in colon tumors werecompared with benign tissue. Normalized PNCK expression levels in thebenign tissues was set equal to 1.0. This analysis demonstrated thatPNCK is expressed in colon tumors at a level that is 5.0-fold lower thanin benign colon tissue (p=0.00031). Further analysis of PNCK expressionas a function of colon tumor grade revealed that PNCK expressioncorrelates negatively with tumor grade with poorly-differentiated tumorsexhibiting lower levels of PNCK expression than moderatelydifferentiated tumors, and moderately-differentiated tumors exhibitinglower levels of PNCK expression than well-differentiated tumors.

Each and every patent, patent application and publication that is citedin the foregoing specification is herein incorporated by reference inits entirety.

While the foregoing specification has been described with regard tocertain preferred embodiments, and many details have been set forth forthe purpose of illustration, it will be apparent to those skilled in theart that the invention may be subject to various modifications andadditional embodiments, and that certain of the details described hereincan be varied considerably without departing from the spirit and scopeof the invention. Such modifications, equivalent variations andadditional embodiments are also intended to fall within the scope of theappended claims.

1. An isolated nucleotide sequence comprising the nucleotide sequence set forth in SEQID No:1.
 2. A An isolated recombinant cell comprising the isolated nucleotide sequence of claim
 1. 3. A vector comprising the isolated nucleotide sequence of claim
 1. 4. An isolated nucleic acid sequence comprising the full complement of the nucleic acid sequence of claim
 1. 5. An isolated mammalian cell comprising the full DNA complement of the nucleic acid sequence of claim
 1. 6. An isolated nucleotide sequence consisting of the nucleotide sequence set forth in SEQID No:
 1. 